|Call||Career Development Fellowship (CDF)|
Rapid detection of Mycobaterium ulcerans infection by recombinase polymerase amplification
This study is to explore the potential of using recombinase polymerase amplification (RPA) as a tool for Buruli ulcer diagnosis. RPA has emerged as a novel isothermal technology for use in molecular diagnosis of infectious diseases. This means that it can be carried out at room temperature in unsophisticated laboratories in West Africa close to patients. The key goal is to develop a diagnostic test for the early detection of Buruli ulcer in symptomatic patients with sufficient positive predictive value to put patients on appropriate treatment.
|Kwame Nkrumah University of Science and Technology||Ghana|
|Ashanti Akim North|
|Ahafo Ano North|
|Upper Denkyira Districts|
|Title||University||Start Date||End Date|
|Cellular immune response to mycobacterium ulcerans infection||Kwame Nkrumah University of Science and Technology||2011-08-01||2015-06-01|
|Type||Name||Title||University||Start Date||End Date|
|Mphil||Hubert S. Ahor||Mr||Kwame Nkrumah University of Science and Technology||2017||2019|
|Mphil||Louis Kyei-Tuffour||Mr||Kwame Nkrumah University of Science and Technology||2018||2020|
|Simpson H et al. Mapping the global distribution of Buruli ulcer: a systematic review with evidence consensus. Lancet Glob Health. 2019 Jul;7(7):e912-e922. doi: 10.1016/S2214-109X(19)30171-8|
|Frimpong M et al. Rapid detection of Mycobacterium ulcerans with isothermal recombinase polymerase amplification assay. PLoS Negl Trop Dis. 2019 Feb 1;13(2):e0007155. doi: 10.1371/journal.pntd.0007155|
|Frimpong, Michael, et al. "Paradoxical reactions in Buruli ulcer after initiation of antibiotic therapy: Relationship to bacterial load." PLoS neglected tropical diseases 13.8 (2019): e0007689|
|Frimpong, M.; Ahor, H.S.; Sakyi, S.A.; Agbavor, B.; Akowuah, E.; Phillips, R.O. Rapid Extraction Method of Mycobacterium ulcerans DNA from Clinical Samples of Suspected Buruli Ulcer Patients. Diagnostics 2019, 9, 204|
|Amoako, Y.A., Frimpong, M., Awuah, D.O. et al. Providing insight into the incubation period of Mycobacterium ulcerans disease: two case reports. J Med Case Reports 13, 218 (2019). https://doi.org/10.1186/s13256-019-2144-2|
Kumasi Centre for Collaborative Research in Tropical Medicine
|Type||Name||Title||University||Start Date||End Date|
|Mphil||Hubert S. Ahor||Mr.||Kwame Nkrumah University of Science and Technology||2017||2019|
|MSc.||Abigail Agbanyo||Ms.||Kwame Nkrumah University of Science and Technology||2017||2019|
|MPhil||Louis Kyei-Tuffour||Mr||Kwame Nkrumah University of Science and Technology||2018||2020|
|MPhil||Venus Boakyewaa Frempong||Ms||Kwame Nkrumah University of Science and Technology||2019||2021|
|Role||Committee/board||Start Date||End Date|
|Kwame Nkrumah University of Science and Technology, Ghana||PhD||2015-06-01|
|Ludwig Maximilian University, Munich, Germany||PGDip||2013-03-01|
Neglected Infectious Diseases (NID) Point-of-care tests Molecular diagnostics
Buruli ulcer is a neglected tropical disease caused by Mycobacterium ulcerans infection that damages the skin and subcutis. It is most prevalent in western and central Africa and Australia. Standard antimicrobial treatment with oral rifampicin 10 mg/kg plus intramuscular streptomycin 15 mg/kg once daily for 8 weeks (RS8) is highly effective, but streptomycin injections are painful and potentially harmful. We aimed to compare the efficacy and tolerability of fully oral rifampicin 10 mg/kg plus clarithromycin 15 mg/kg extended release once daily for 8 weeks (RC8) with that of RS8 for treatment of early Buruli ulcer lesions.
We did an open-label, non-inferiority, randomised (1:1 with blocks of six), multicentre, phase 3 clinical trial comparing fully oral RC8 with RS8 in patients with early, limited Buruli ulcer lesions. There were four trial sites in hospitals in Ghana (Agogo, Tepa, Nkawie, Dunkwa) and one in Benin (Pobè). Participants were included if they were aged 5 years or older and had typical Buruli ulcer with no more than one lesion (caterories I and II) no larger than 10 cm in diameter. The trial was open label, and neither the investigators who took measurements of the lesions nor the attending doctors were masked to treatment assignment. The primary clinical endpoint was lesion healing (ie, full epithelialisation or stable scar) without recurrence at 52 weeks after start of antimicrobial therapy. The primary endpoint and safety were assessed in the intention-to-treat population. A sample size of 332 participants was calculated to detect inferiority of RC8 by a margin of 12%. This study was registered with ClinicalTrials.gov, NCT01659437.
Between Jan 1, 2013, and Dec 31, 2017, participants were recruited to the trial. We stopped recruitment after 310 participants. Median age of participants was 14 years (IQR 10–29) and 153 (52%) were female. 297 patients had PCR-confirmed Buruli ulcer; 151 (51%) were assigned to RS8 treatment, and 146 (49%) received oral RC8 treatment. In the RS8 group, lesions healed in 144 (95%, 95% CI 91 to 98) of 151 patients, whereas lesions healed in 140 (96%, 91 to 99) of 146 patients in the RC8 group. The difference in proportion, −0·5% (–5·2 to 4·2), was not significantly greater than zero (p=0·59), showing that RC8 treatment is non-inferior to RS8 treatment for lesion healing at 52 weeks. Treatment-related adverse events were recorded in 20 (13%) patients receiving RS8 and in nine (7%) patients receiving RC8. Most adverse events were grade 1–2, but one (1%) patient receiving RS8 developed serious ototoxicity and ended treatment after 6 weeks. No patients needed surgical resection. Four patients (two in each study group) had skin grafts.
Fully oral RC8 regimen was non-inferior to RS8 for treatment of early, limited Buruli ulcer and was associated with fewer adverse events. Therefore, we propose that fully oral RC8 should be the preferred therapy for early, limited lesions of Buruli ulcer.
Isothermal amplification techniques such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) for diagnosing Buruli ulcer, a necrotic skin disease caused by Mycobacterium ulcerans, have renewed hope for the molecular diagnosis of clinically suspected Buruli ulcer cases in endemic districts. If these techniques are applied at district-level hospitals or clinics, they will help facilitate early case detection with prompt treatment, thereby reducing disability and associated costs of disease management. The accuracy as well as the application of these molecular techniques at point of need is dependent on simple and fast DNA extraction. We have modified and tested a rapid extraction protocol for use with an already developed recombinase polymerase amplification assay. The entire procedure from "sample in, extraction and DNA amplification" was conducted in a mobile suitcase laboratory within 40 min. The DNA extraction procedure was performed within 15 min, with only two manipulation/pipetting steps needed. The diagnostic sensitivity and specificity of this extraction protocol together with M. ulcerans RPA in comparison with standard DNA extraction with real-time PCR was 87% (n = 26) and 100% (n = 13), respectively. We have established a simple, fast and efficient protocol for the extraction and detection of M. ulcerans DNA in clinical samples that is adaptable to field conditions.
Buruli ulcer (BU) is a chronic ulcerating tropical skin disease known to particularly affect vulnerable populations. Without early detection and effective treatment it can lead to disfigurement, disability and stigma. In order to improve outcomes, we need to understand what factors prevent patients from accessing and completing treatment, however these factors are often not well understood. Factors considered to potentially affect treatment completion include access to care and type of treatment. In this study we analysed data available from clinical records of patients treated in Ghana to identify whether type of treatment and common patient characteristics were associated with treatment completion. We found that treatment completion was higher in patients who took a newly introduced oral treatment compared to those who took the traditional injectable treatment. We did not find a difference in treatment completion between patients living close to the clinic and those living further away, however we found that those living further were more likely to present with more advanced disease. The results from this study suggest that management for patients living far from care needs to be improved. The newly recommended oral treatment makes it feasible to provide care away from health centres and the improved treatment completion seen in this study supports its use. However, further research should be conducted to determine how fully community based care can best be provided.
Background: Buruli ulcer caused by Mycobacterium ulcerans is endemic in parts of West Africa and is most prevalent among the 5-15 years age group; Buruli ulcer is uncommon among neonates. The mode of transmission and incubation period of Buruli ulcer are unknown. We report two cases of confirmed Buruli ulcer in human immunodeficiency virus-unexposed, vaginally delivered term neonates in Ghana.
Case presentation: Patient 1: Two weeks after hospital delivery, a baby born to natives of the Ashanti ethnic group of Ghana was noticed by her mother to have a papule with associated edema on the right anterior chest wall and neck that later ulcerated. There was no restriction of neck movements. The diagnosis of Buruli ulcer was confirmed on the basis of a swab sample that had a positive polymerase chain reaction result for the IS2404 repeat sequence of M. ulcerans. Patient 2: This patient, from the Ashanti ethnic group in Ghana, had the mother noticing a swelling in the baby's left gluteal region 4 days after birth. The lesion progressively increased in size to involve almost the entire left gluteal region. Around the same time, the mother noticed a second, smaller lesion on the forehead and left side of neck. The diagnosis of Buruli ulcer was confirmed by polymerase chain reaction when the child was aged 4 weeks. Both patients 1 and 2 were treated with oral rifampicin and clarithromycin at recommended doses for 8 weeks in addition to appropriate daily wound dressing, leading to complete healing. Our report details two cases of polymerase chain reaction-confirmed Buruli ulcer in children whose lesions appeared at ages 14 and 4 days, respectively. The mode of transmission of M. ulcerans infection is unknown, so the incubation period is difficult to estimate and is probably dependent on the infective dose and the age of exposure. In our study, lesions appeared 4 days after birth in patient 2. Unless the infection was acquired in utero, this would be the shortest incubation period ever recorded.
Conclusions: Buruli ulcer should be included in the differential diagnosis of neonates who present with characteristic lesions. The incubation period of Buruli ulcer in neonates is probably shorter than is reported for adults.
Introduction: Buruli ulcer (BU) caused by Mycobacterium ulcerans is effectively treated with rifampicin and streptomycin for 8 weeks but some lesions take several months to heal. We have shown previously that some slowly healing lesions contain mycolactone suggesting continuing infection after antibiotic therapy. Now we have determined how rapidly combined M. ulcerans 16S rRNA reverse transcriptase / IS2404 qPCR assay (16S rRNA) became negative during antibiotic treatment and investigated its influence on healing.
Methods: Fine needle aspirates and swab samples were obtained for culture, acid fast bacilli (AFB) and detection of M. ulcerans 16S rRNA and IS2404 by qPCR (16S rRNA) from patients with IS2404 PCR confirmed BU at baseline, during antibiotic and after treatment. Patients were followed up at 2 weekly intervals to determine the rate of healing. The Kaplan-Meier survival analysis was used to analyse the time to clearance of M. ulcerans 16S rRNA and the influence of persistent M ulcerans 16S rRNA on time to healing. The Mann Whitney test was used to compare the bacillary load at baseline in patients with or without viable organisms at week 4, and to analyse rate of healing at week 4 in relation to detection of viable organisms.
Results: Out of 129 patients, 16S rRNA was detected in 65% of lesions at baseline. The M. ulcerans 16S rRNA remained positive in 78% of patients with unhealed lesions at 4 weeks, 52% at 8 weeks, 23% at 12 weeks and 10% at week 16. The median time to clearance of M. ulcerans 16S rRNA was 12 weeks. BU lesions with positive 16S rRNA after antibiotic treatment had significantly higher bacterial load at baseline, longer healing time and lower healing rate at week 4 compared with those in which 16S rRNA was not detected at baseline or had become undetectable by week 4.
Conclusions: Current antibiotic therapy for BU is highly successful in most patients but it may be possible to abbreviate treatment to 4 weeks in patients with a low initial bacterial load. On the other hand persistent infection contributes to slow healing in patients with a high bacterial load at baseline, some of whom may need antibiotic treatment extended beyond 8 weeks. Bacterial load was estimated from a single sample taken at baseline. A better estimate could be made by taking multiple samples or biopsies but this was not ethically acceptable.
Background: We investigated the relationship between bacterial load in Buruli ulcer (BU) lesions and the development of paradoxical reaction following initiation of antibiotic treatment.
Methods: This was a longitudinal study involving BU patients from June 2013 to June 2017. Fine needle aspirates (FNA) and swab samples were obtained to establish the diagnosis of BU by PCR. Additional samples were obtained at baseline, during and after treatment (if the lesion had not healed) for microscopy, culture and combined 16S rRNA reverse transcriptase/ IS2404 qPCR assay. Patients were followed up at regular intervals until complete healing.
Results: Forty-seven of 354 patients (13%) with PCR confirmed BU had a PR, occurring between 2 and 42 (median 6) weeks after treatment initiation. The bacterial load, the proportion of patients with positive M. ulcerans culture (15/34 (44%) vs 29/119 (24%), p = 0.025) and the proportion with positive microscopy results (19/31 (61%) vs 28/90 (31%), p = 0.003) before initiation of treatment were significantly higher in the PR compared to the no PR group. Plaques (OR 5.12; 95% CI 2.26-11.61; p<0.001), oedematous (OR 4.23; 95% CI 1.43-12.5; p = 0.009) and category II lesions (OR 2.26; 95% CI 1.14-4.48; p = 0.02) were strongly associated with the occurrence of PR. The median time to complete healing (28 vs 13 weeks, p <0.001) was significantly longer in the PR group.
Conclusions: Buruli ulcer patients who develop PR are characterized by high bacterial load in lesion samples taken at baseline and a higher rate of positive M. ulcerans culture. Occurrence of a PR was associated with delayed healing.
Background: Antibiotic treatment proved itself as the mainstay of treatment for Buruli ulcer disease. This neglected tropical disease is caused by Mycobacterium ulcerans. Surgery persists as an adjunct therapy intended to reduce the mycobacterial load. In an earlier clinical trial, patients benefited from delaying the decision to operate. Nevertheless, the rate of surgical interventions differs highly per clinic.Methods: A retrospective study was conducted in six different Buruli ulcer (BU) treatment centers in Benin and Ghana. BU patients clinically diagnosed between January 2012 and December 2016 were included and surgical interventions during the follow-up period, at least one year after diagnosis, were recorded. Logistic regression analysis was carried out to estimate the effect of the treatment center on the decision to perform surgery, while controlling for interaction and confounders.Results: A total of 1193 patients, 612 from Benin and 581 from Ghana, were included. In Benin, lesions were most frequently (42%) categorized as the most severe lesions (WHO criteria, category III), whereas in Ghana lesions were most frequently (44%) categorized as small lesions (WHO criteria, category I). In total 344 (29%) patients received surgical intervention. The percentage of patients receiving surgical intervention varied between hospitals from 1.5% to 72%. Patients treated in one of the centers in Benin were much more likely to have surgery compared to the clinic in Ghana with the lowest rate of surgical intervention (RR = 46.7 CI 95% [17.5-124.8]). Even after adjusting for confounders (severity of disease, age, sex, limitation of movement at joint at time of diagnosis, ulcer and critical sites), rates of surgical interventions varied highly. Conclusion: The decision to perform surgery to reduce the mycobacterial load in BU varies highly per clinic. Evidence based guidelines are needed to guide the role of surgery in the treatment of BU.
|Prof. Richard Phillips||Ghana||Kumasi Centre for Collaborative Research|
|Dr. Michael Frimpong||Ghana||Kumasi Centre for Collaborative Research|