Dr
Michael Frimpong
Project Title
Rapid detection of Mycobaterium ulcerans infection by recombinase polymerase amplification
EDCTP Project
TMA2015CDF979
EDCTP Program
EDCTP2
EDCTP Project Call
Career Development Fellowship (CDF)
Project Objectives
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.
Study Design
Cross-sectional study
Project Summary
There are no primary measures to prevent people from contracting Buruli ulcer, mainly due to poor understanding of its epidemiology. The current control strategy emphasizes early diagnosis and prompt treatment, with the goal of avoiding the complications associated with advanced stages of the disease. There is no diagnostic test for the disease appropriate for use at the primary health care level where most cases are detected and treated. Diagnosis based on clinical signs is unrealiable in inexperienced hands and complicated by infections that have similar presentations. The aim of this study is to develop and evaluate the use of recombinase polymerase amplification assay for the detection of Mycobacterium ulcerans the causative agent of Buruli ulcer at the point of patient care.
Host Organisation
| Department | Institution | Country |
|---|---|---|
| Kumasi Centre for Collaborative Research in Tropical Medicine | Kwame Nkrumah University of Science and Technology | Ghana |
Sites
Results & Outcomes
Current Organisation
Kumasi Centre for Collaborative Research in Tropical Medicine
Current Job Title
Research Fellow
Biography
Publications
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.
Non-typhoidal Salmonella (NTS) cause the majority of bloodstream infections in Ghana, however the mode of transmission and source of invasive NTS in Africa are poorly understood. This study compares NTS from water sources and invasive bloodstream infections in rural Ghana. Blood from hospitalised, febrile children and samples from drinking water sources were analysed for Salmonella spp. Strains were serotyped to trace possible epidemiological links between human and water-derived isolates.. Antibiotic susceptibility testing was performed, In 2720 blood culture samples, 165 (6%) NTS were isolated. S. Typhimurium (70%) was the most common serovar followed by S. Enteritidis (8%) and S. Dublin (8%). Multidrug resistance (MDR) was found in 95 (58%) NTS isolates, including five S. Enteritidis.
Author summary
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 can cause disfigurement and long-term loss of function. It is underdiagnosed and under-reported, and its current distribution is unclear. We aimed to synthesise and evaluate data on Buruli ulcer prevalence and distribution.
METHODS: We did a systematic review of Buruli ulcer prevalence and used an evidence consensus framework to describe and evaluate evidence for Buruli ulcer distribution worldwide. We searched PubMed and Web of Science databases from inception to Aug 6, 2018, for records of Buruli ulcer and Mycobacterium ulcerans detection, with no limits on study type, publication date, participant population, or location. English, French, and Spanish language publications were included. We included population-based surveys presenting Buruli ulcer prevalence estimates, or data that allowed prevalence to be estimated, in the systematic review. We extracted geographical data on the occurrence of Buruli ulcer cases and M ulcerans detection from studies of any type for the evidence consensus framework; articles that did not report original data were excluded. For the main analysis, we extracted prevalence estimates from included surveys and calculated 95% CIs using Byar's method. We included occurrence records, reports to WHO and the Global Infectious Diseases and Epidemiology Network, and surveillance data from Buruli ulcer control programmes in the evidence consensus framework to grade the strength of evidence for Buruli ulcer endemicity. This study is registered with PROSPERO, number CRD42018116260.
FINDINGS: 2763 titles met the search criteria. We extracted prevalence estimates from ten studies and occurrence data from 208 studies and five unpublished surveillance datasets. Prevalence estimates within study areas ranged from 3·2 (95% CI 3·1-3·3) cases per 10 000 population in Côte d'Ivoire to 26·9 (23·5-30·7) cases per 10 000 population in Benin. There was evidence of Buruli ulcer in 32 countries and consensus on presence in 12.
INTERPRETATION: The global distribution of Buruli ulcer is uncertain and potentially wider than currently recognised. Our findings represent the strongest available evidence on Buruli ulcer distribution so far and have many potential applications, from directing surveillance activities to informing burden estimates
BACKGROUND: Access to an accurate diagnostic test for Buruli ulcer (BU) is a research priority according to the World Health Organization. Nucleic acid amplification of insertion sequence IS2404 by polymerase chain reaction (PCR) is the most sensitive and specific method to detect Mycobacterium ulcerans (M. ulcerans), the causative agent of BU. However, PCR is not always available in endemic communities in Africa due to its cost and technological sophistication. Isothermal DNA amplification systems such as the recombinase polymerase amplification (RPA) have emerged as a molecular diagnostic tool with similar accuracy to PCR but having the advantage of amplifying a template DNA at a constant lower temperature in a shorter time. The aim of this study was to develop RPA for the detection of M. ulcerans and evaluate its use in Buruli ulcer disease.
METHODOLOGY AND PRINCIPAL FINDINGS: A specific fragment of IS2404 of M. ulcerans was amplified within 15 minutes at a constant 42°C using RPA method. The detection limit was 45 copies of IS2404 molecular DNA standard per reaction. The assay was highly specific as all 7 strains of M. ulcerans tested were detected, and no cross reactivity was observed to other mycobacteria or clinically relevant bacteria species. The clinical performance of the M. ulcerans (Mu-RPA) assay was evaluated using DNA extracted from fine needle aspirates or swabs taken from 67 patients in whom BU was suspected and 12 patients with clinically confirmed non-BU lesions. All results were compared to a highly sensitive real-time PCR. The clinical specificity of the Mu-RPA assay was 100% (95% CI, 84-100), whiles the sensitivity was 88% (95% CI, 77-95).
CONCLUSION: The Mu-RPA assay represents an alternative to PCR, especially in areas with limited infrastructure.
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.
BACKGROUND: Non-typhoidal Salmonella (NTS) cause the majority of bloodstream infections in Ghana, however the mode of transmission and source of invasive NTS in Africa are poorly understood. This study compares NTS from water sources and invasive bloodstream infections in rural Ghana.
METHODS: Blood from hospitalised, febrile children and samples from drinking water sources were analysed for Salmonella spp. Strains were serotyped to trace possible epidemiological links between human and water-derived isolates.. Antibiotic susceptibility testing was performed, RESULTS: In 2720 blood culture samples, 165 (6%) NTS were isolated. S. Typhimurium (70%) was the most common serovar followed by S. Enteritidis (8%) and S. Dublin (8%). Multidrug resistance (MDR) was found in 95 (58%) NTS isolates, including five S. Enteritidis. One S. Typhimurium showed reduced fluroquinolone susceptibility. In 511 water samples, 19 (4%) tested positive for S. enterica with two isolates being resistant to ampicillin and one isolate being resistant to cotrimoxazole. Serovars from water samples were not encountered in any of the clinical specimens.
CONCLUSION: Water analyses demonstrated that common drinking water sources were contaminated with S. enterica posing a potential risk for transmission. However, a link between S. enterica from water sources and patients could not be established, questioning the ability of water-derived serovars to cause invasive bloodstream infections
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.
Background: Buruli ulcer is a disease of the skin and soft tissues caused by infection with a slow growing pathogen, Mycobacterium ulcerans. A vaccine for this disease is not available but M. ulcerans possesses a giant plasmid pMUM001 that harbours the polyketide synthase (PKS) genes encoding a multi-enzyme complex needed for the production of its unique lipid toxin called mycolactone, which is central to the pathogenesis of Buruli ulcer. We have studied the immunogenicity of enzymatic domains in humans with M. ulcerans disease, their contacts, as well as non-endemic areas controls.
Methods: Between March 2013 and August 2015, heparinized whole blood was obtained from patients confirmed with Buruli ulcer. The blood samples were diluted 1 in 10 in Roswell Park Memorial Institute (RPMI) medium and incubated for 5 days with recombinant mycolactone PKS domains and mycolyltransferase antigen 85A (Ag85A). Blood samples were obtained before and at completion of antibiotic treatment for 8 weeks and again 8 weeks after completion of treatment. Supernatants were assayed for interferon-γ (IFN-γ) and interleukin-5 (IL-5) by enzyme-linked immunosorbent assay. Responses were compared with those of contacts and non-endemic controls.
Results: More than 80% of patients and contacts from endemic areas produced IFN-γ in response to all the antigens except acyl carrier protein type 3 (ACP3) to which only 47% of active Buruli ulcer cases and 71% of contacts responded. The highest proportion of responders in cases and contacts was to load module ketosynthase domain (Ksalt) (100%) and enoylreductase (100%). Lower IL-5 responses were induced in a smaller proportion of patients ranging from 54% after ketoreductase type B stimulation to only 21% with ketosynthase type C (KS C). Among endemic area contacts, the, highest proportion was 73% responding to KS C and the lowest was 40% responding to acyltransferase with acetate specificity type 2. Contacts of Buruli ulcer patients produced significantly higher IFN-γ and IL-5 responses compared with those of patients to PKS domain antigens and to mycolyltransferase Ag85A of M. ulcerans. There was low or no response to all the antigens in non-endemic areas controls. IFN-γ and IL-5 responses of patients improved after treatment when compared to baseline results.
Discussion: The major response to PKS antigen stimulation was IFN-γ and the strongest responses were observed in healthy contacts of patients living in areas endemic for Buruli ulcer. Patients elicited lower responses than healthy contacts, possibly due to the immunosuppressive effect of mycolactone, but the responses were enhanced after antibiotic treatment. A vaccine made up of the most immunogenic PKS domains combined with the mycolyltransferase Ag85A warrants further investigation.