Project

Medical treatment of Eumycetoma

Goal: To discover new drugs for the treatment of Eumycetoma

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Ahmed Hassan Fahal
added a research item
ABSTRACT Late presentation of mycetoma patients to attain medical care is one of the most important prognostic factors that is strongly associated with a poor treatment outcome and high amputation rate. The present study explored the prevalence and causes of the late presentation of mycetoma patients presenting to the Mycetoma Research Centre (MRC), Soba University Hospital. It included 288 patients with confirmed mycetoma. The data were collected using a pre-designed structured questionnaire by direct personal interview. In this study, 138 patients (48%) were less than 30 years of age at presentation, and 228 patients (79%) were males. The patients were from different parts of the country, but most of them were from El-Gezira (n=85, 29.5%) and Sennar (n=38, 13.2%) States. Most of the patients (n=180, 62.5%) had either only basic primary education or none. The study showed that 118 (40.9%) patients were severely affected by the disease, had dropped education or job and were unable to perform their routine daily activities. Most of the patients 209 (72.6%) had disease duration of more than one year at presentation while only 79 (27.4%) patients had a disease duration of less than one year. Due to the mycetoma painless nature, 198 (94.7%) patients ignored the disease initially, 83 (39.2%) of them attributed the late presentation to financial reasons, and geographical distance was a cause in 43 (20.6%) patients. One hundred and nineteen (56.9%) patients presented to their local health facilities, 53 (44.5%) of them received inappropriate treatment without being diagnosed as mycetoma, and 66 (55.5%) patients received insufficient treatment despite being diagnosed as mycetoma. The study showed a statistically significant association between the patients’ age, level of education, marital status, occupation and the late presentation. From the obtained results, it is recommended that more efforts to raise disease awareness and advocacy on early presentation, diagnosis and treatment among both the population and health care providers are needed in particularly in endemic areas. Training of health care providers on early case detection and management in mycetoma endemic areas is mandatory. Improvement of the local health and medical facilities in endemic areas is essential for early mycetoma diagnosis and treatment; and to reduce the geographical distance between the affected villages and the tertiary centres.
Ahmed Hassan Fahal
added a research item
A 57 year-old male, from central Sudan, presented to the Mycetoma Research Centre (MRC), Khartoum, Sudan, with a slowly progressive right inguino-scrotal swelling of 35 years duration. He also reported local scrotal pain and itching. The swelling was first noted in 1982 as a small painless mass in the lower inner quadrant of the right gluteal region. By 2005, it progressively increased in size and a purulent discharge containing black grains appeared. The patient did not recall any history of local trauma. He underwent surgical excision under local anaesthesia at a regional hospital. Two years later he developed local recurrence that extended to the perineal area just below the scrotum that started to discharge pus and black grains. He underwent further surgical excision under spinal anaesthesia. However, following these surgical excisions, no tissue diagnosis was established, and no treatment was given. In 2010, he presented to the MRC complaining of right hemiscrotal painless swelling of six months duration. Clinical examination revealed a mass measuring 5×4 cm, with multiple sinuses discharging pus and black grains; it was soft in consistency and was attached to the skin and deep structures. The diagnosis of Madurella mycetomatis eumycetoma was established by cytological examination of fine needle aspirate from the lesion. He was commenced on 200mg ketoconazole twice daily orally and folic acid 5 mg once a day orally. Two months later he underwent wide local excision at the MRC under spinal anaesthesia. He remained inpatient for 45 days for daily wound dressing and was discharged in good condition. He then had reconstructive plastic surgery to cover the open wound by a skin graft. A follow-up ultrasound examination three months later identified local recurrence. Sadly, he stopped taking his medications due to social reasons. In 2013 he presented to the MRC with local recurrence, and the lesion ultrasound examination revealed right testicular invasion. He was commenced on 200 mg itraconazole twice daily orally, and folic acid tabs 5 mg once a day; however, the swelling did not regress in size with treatment. Four years later, in 2017, the patient noticed a rapid increase in the swelling, which extended to the right inguinal region. Seven months prior to his presentation, he developed scrotal pain and itching, which was of low grade and mainly nocturnally. He had no constitutional symptoms. The patient is diabetic on 500mg metformin twice daily for four years. He was not on other long-term medication. He has a family history of mycetoma; his brother had a right foot eumycetoma. He worked as a cashier, married with 12 children and the youngest is 12-year-old. On clinical examination, the patient looked well, neither pale, jaundiced, nor cyanosed. He had no
Sami Ahmed Khalid
added 2 research items
Plasmodium falciparum is the most serious health threat in Sub-Saharan Africa1. This situation is further aggravated by the resistance to currently known antimalarials coupled with the lack of an effective vaccine. Therefore, there is an urgent need to discover new viable biochemical targets and biologically active compounds. The discovery of a type II fatty acid biosynthesis pathway (FAS II) in P. falciparum, particularly, the enoyl-ACP reductase (PfENR), which catalyses the rate limiting step in each elongation circle, has been recognized and validated as an important target2,3. The present work capitalizes on the discovery of new antimalarial molecules based on PfENR inhibition. The alcoholic extracts of five plants commonly used in the Sudanese traditional medicine to treat malaria exhibited certain degree of inhibition of PfENR in concentrations less than 250 μg/mL. Bioactivity-guided fractionation revealed that the most potent inhibitors of PfENR were the ethyl acetate fractions of Acacia nilotica and Khaya senegalensis stem barks followed by their water residue and finally the Ziziphus spina-christi root bark with IC50 values of 0.87, 3.35, 6.33, 11.68 and 15.62μg/mL, respectively. The very prominent activity of the ethyl acetate fraction of Acacia nilotica had encouraged us to further analyze it in order to isolate bioactive compound(s) associated with the aforementioned activity. References 1. World malaria report: 2012. WHO. 2. Freundlich, J. S.; Anderson, J. W.; Sarantakis, D.; Shieh, H.-M.; Yu, M.; Valderramos, J.-C.; Lucumi, et al., Bioorg. Med. Chem. Lett. 2005, 15, 5247-52. 3. Perozzo, R.; Kuo, M.; Sidhu, A. B. S.; Valiyaveettil, J. T.; Bittman, R.; Jacobs, W. R.; et al., Biol. Chem. 2002, 277, 13106 - 14.
Sami Ahmed Khalid
added an update
More fractions and pure compounds are currently under investigation. It is anticipated to present the outcomes of this research in a congress in France during July this year. Further results will be presented in the 65th Annual Meeting of the GA in Basel, Switzerland in September 2017.
 
Ahmed Hassan Fahal
added 2 research items
The current treatment of eumycetoma utilizing ketoconazole is unsatisfactory because of high recurrence rates, which often leads to complications and unnecessary amputations, and its comparatively high cost in endemic areas. Hence, an effective and affordable drug is required to improve therapeutic outcome. E1224 is a potent orally available, broad-spectrum triazole currently being developed for the treatment of Chagas disease. E1224 is a prodrug that is rapidly converted to ravuconazole. Plasma levels of E1224 are low and transient, and its therapeutically active moiety, ravuconazole is therapeutically active. In the present study, the in vitro activity of ravuconazole against Madurella mycetomatis, the most common etiologic agent of eumycetoma, was evaluated and compared to that of ketoconazole and itraconazole. Ravuconazole showed excellent activity with MICs ranging between #0.002 and 0.031 mg/ml, which were significantly lower than the MICs reported for ketoconazole and itraconazole. On the basis of our findings, E1224 with its resultant active moiety, ravuconazole, could be an effective and affordable therapeutic option for the treatment of eumycetoma.
Abstract Eumycetoma is a chronic progressive disabling and destructive inflammatory disease which is commonly caused by the fungus Madurella mycetomatis. It is characterized by the formation of multiple discharging sinuses. It is usually treated by antifungal agents but it is assumed that the therapeutic efficiency of these agents is reduced by the co-existence of Staphylococcus aureus co-infection developing in these sinuses. This prospective study was conducted to investigate the safety, efficacy and clinical outcome of combined antibiotic and antifungal therapy in eumycetoma patients with superimposed Staphylococcus aureus infection. The study enrolled 337 patients with confirmed M. mycetomatis eumycetoma and S. aureus co-infection. Patients were allocated into three groups; 142 patients received amoxicillinclavulanic acid and ketoconazole, 93 patients received ciprofloxacin and ketoconazole and 102 patients received ketoconazole only. The study showed that, patients who received amoxicillin-clavulanic acid and ketoconazole treatment had an overall better clinical outcome compared to those who had combined ciprofloxacin and ketoconazole or to those who received ketoconazole only. In this study, 60.6% of the combined amoxicillin-clavulanic acid/ketoconazole group showed complete or partial clinical response to treatment compared to 30.1% in the ciprofloxacin/ketoconazole group and 36.3% in the ketoconazole only group. The study also showed that 64.5% of the patients in the ciprofloxacin/ketoconazole group and 59.8% in the ketoconazole only group had progressive disease and poor outcome. This study showed that the combination of amoxicillin-clavulanic acid and ketoconazole treatment is safe and offers good clinical outcome and it is therefore recommended to treat eumycetoma patients with Staphylococcus aureus co-infection.
Ahmed Hassan Fahal
added a research item
Objective: To determine if combination therapy would improve therapeutic outcome in eumycetoma caused by Madurella mycetomatis. Methods: Survival, colony forming units (CFU), melanisation and histopathology in M. mycetomatis infected Galleria mellonella larvae treated with amphotericin B, itraconazole, terbinafine or combinations thereof were determined. Results: Compared to larvae treated with 5% glucose, enhanced survival was obtained when M. mycetomatis-infected larvae were treated with amphotericin B, but not when they were treated with itraconazole or terbinafine. Combination therapy did not increase survival compared to 5% glucose treated larvae, itraconazole treated larvae or terbinafine treated larvae. Compared to amphotericin B monotreatment a significant decrease in survival was noted when this therapy was combined with either itraconazole or terbinafine. CFU, melanisation and histopathology did not differ between monotherapy, combination therapy or 5% glucose treated larvae. Conclusions: Combining different classes of antifungal agents did not enhance the survival of M. mycetomatis infected G. mellonella larvae. Instead of improving the therapeutic outcome, combining either itraconazole or terbinafine with amphotericin B resulted in significantly lower survival rates of infected larvae than amphotericin B monotherapy. This experimental study does not provide support for the use of combined amphotericin B and itraconazole, combined itraconazole and terbinafine or combined terbinafine and amphotericin B, and should be confirmed in other animal models. This article is protected by copyright. All rights reserved.
Sami Ahmed Khalid
added 5 research items
Madurella mycetomatis is one of the most prevalent causative agents of black-grain eumycetoma. Infection caused by this fungus is notoriously difficult to treat due to absence of adequate therapy and frequently leads to surgical excision of the infected limb. Prolonged follow-up after surgery with currently available antifungals, however, might improve the clinical outcome [1]. Nevertheless, the increasing recurrence and resistance of these pathogenic organisms to existing antifungals warrant the development a reproducible in vitro assay for discovery of novel bioactive agents [2]. The new 96-well microplate’s assay based on resazurin dye, which was developed by our group to assess the antifungal activity against Madurella mycetomatis [3] is presently applied to 10 essential oils. GC/MS analysis was carried out with an HP 5890 Series II gas chromatograph (FID) and the identification of the compounds was based on comparisons with published MS data and a computer library search. In vitro, M. mycetomatis appears to be highly susceptible to essential oils, hence the MICs of the essential oils ranged between 0.625 - 2.5 mg/mL, while the standard antifungal, itraconazole exhibited MIC of 0.354 µM. More than 300 mono- and sesquiterpenes were identified and a correlation between the bioactivity and the identified compound has been hypothesised. References: 1. van de Sande WWJ. Global Burden of Human Mycetoma: A systematic review and meta-analysis. PLoS Negl Trop Dis 2013; 7: e2550. doi:10.1371/journal.pntd.0002550. 2. van Belkum A, Fahal AH, van de Sande WWJ. In vitro susceptibility of Madurella mycetomatis to posaconazole and terbinafine. Antimicrob Agents Chemother 2011; 55: 1771–1773 3. Khalid SA. Development of microtiter plate-based method for the determination of the MIC of antimycetomal agents against Madurella mycetomatis. II ResNet NPND workshop on natural products against neglected diseases, Nov. 25-28th, 2014, Rio de Janeiro, Brazil.
The decoction of different parts of Nauclea latifolia Smith (Rubiaceae) is commonly employed in the African traditional medicine for the treatment of several diseases, such as malaria, gastrointestinal tract disorders, and as antimicrobial [1]. It has already been demonstrated that N. latifolia is very efficient in the biosynthesis of β-carboline alkaloids besides other secondary metabolites [2]. In our quest to identify a new antifungal agent from natural source we screened the crude extract of the fruits, root bark, leaves and stem bark of Nauclea latifolia in vitro against Madurella mycetomatis, the most common eumycetoma causative organism. Following bioactivity guided fractionation while employing the newly developed resazurin assay in 96-microplate [3] a promising inhibitory activity emerged against M. mycetomatis ranging between 625 and 39.1 µg/mL. This attempt has eventually resulted in the isolation of the β-carboline alkaloid, strictosamide (1), which exhibited a MIC of 3.91 µg/mL while ketoconazole, the positive control showed MIC of 0.25 µg/mL. It is pertinent to note that strictosamide has already exhibited antiparasitic activity against a number of neglected diseases [2]. References 1. Anowi C.F, Cardinal N.C, Ezugwu C.O, Anastasia U, Utoh-Nedosa U.A, Antimicrobial properties of the chloroform extract of the stem bark of Nauclea latifolia, Int J Pharm Pharm Sci, 4, (2), 744-750. 2. Khalid SA. Natural product-based drug discovery against neglected diseases with special reference to African natural resources. In: Chibale K, Devis-Coleman M, Masimirembwa C, editors. Drug discovery in Africa. Berlin Heidelberg: Springer; 2012: 211-237. 3. Khalid SA. Development of microtiter plate-based method for the determination of the MIC of antimycetomal agents against Madurella mycetomatis. II ResNet NPND workshop on natural products against neglected diseases, Nov. 25-28th, 2014, Rio de Janeiro, Brazil.
Sami Ahmed Khalid
added a research item
Madurella mycetomatis is one of the most prevalent causative agents of black-grain eumycetoma. Infection caused by this fungus is notoriously difficult to treat due to absence of adequate therapy and frequently leads to surgical excision of the infected limb. Prolonged follow-up after surgery with currently available antifungals, however, might improve the clinical outcome [1]. Nevertheless, the increasing recurrence and resistance of these pathogenic organisms to existing antifungals warrant the development a reproducible in vitro assay for discovery of novel bioactive agents [2]. The new 96-well microplate's assay based on resazurin dye, which was developed by our group to assess the antifungal activity against Madurella mycetomatis [3] is presently applied to 10 essential oils. GC/MS analysis was carried out with an HP 5890 Series II gas chromatograph (FID) and the identification of the compounds was based on comparisons with published MS data and a computer library search. In vitro, M. mycetomatis appears to be highly susceptible to essential oils, hence the MICs of the essential oils ranged between 0.625 – 2.5 mg/mL, while the standard antifungal, itraconazole exhibited MIC of 0.354 µM. More than 300 mono- and sesquiterpenes were identified and a correlation between the bioactivity and the identified compound has been hypothesised. References: [1] van de Sande WWJ. Global Burden of Human Mycetoma: A systematic review and meta-analysis. PLoS Negl Trop Dis 2013; 7: e2550. doi:10.1371/journal.pntd.0002550. [2] van Belkum A, Fahal AH, van de Sande WWJ. In vitro susceptibility of Madurella mycetomatis to posaconazole and terbinafine. Antimicrob Agents Chemother 2011; 55: 1771 – 1773 [3] Khalid SA. Development of microtiter plate-based method for the determination of the MIC of antimycetomal agents against Madurella mycetomatis. II ResNet NPND Workshop on Natural Products Against Neglected Diseases, Nov. 25 – 28th, 2014, Rio de Janeiro, Brazil.
Lamis Elkheir
added a project goal
To discover new drugs for the treatment of Eumycetoma