Background 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. MethodsWe 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.
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/ IS 2404 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. Trial registration ClinicalTrials.gov NCT02153034 .
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 IS 2404 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 IS 2404 of M . ulcerans was amplified within 15 minutes at a constant 42°C using RPA method. The detection limit was 45 copies of IS 2404 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.
BackgroundBuruli 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.MethodsBetween 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.ResultsMore 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.DiscussionThe 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.
Yaws is a skin debilitating disease caused by Treponema pallidum subspecies pertenue with most cases reported in children. World Health Organization (WHO) aims at total eradication of this disease through mass treatment of suspected cases followed by an intensive follow-up program. However, effective diagnosis is pivotal in the successful implementation of this control program. Recombinase polymerase amplification (RPA), an isothermal nucleic acid amplification technique offers a wider range of differentiation of pathogens including those isolated from chronic skin ulcers with similar characteristics such as Haemophilus ducreyi (H. ducreyi). We have developed a RPA assay for the simultaneous detection of Treponema pallidum (T. pallidum) and H. ducreyi (TPHD-RPA). The assay demonstrated no cross-reaction with other pathogens and enable detection of T. pallidum and H. ducreyi within 15 min at 42 °C. The RPA assay was validated with 49 clinical samples from individuals confirmed to have yaws by serological tests. Comparing the developed assay with commercial multiplex real-time PCR, the assay demonstrated 94% and 95% sensitivity for T. pallidum and H. ducreyi, respectively and 100% specificity. This simple novel TPHD-RPA assay enables the rapid detection of both T. pallidum and H. ducreyi in yaws-like lesions. This test could support the yaws eradication efforts by ensuring reliable diagnosis, to enable monitoring of program success and planning of follow-up interventions at the community level.
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.
BackgroundBuruli ulcer is a chronic ulcerating skin condition, with the highest burden found in Central and West Africa where it disproportionately affects the most vulnerable populations. Treatment is demanding, comprising eight-weeks of daily antibiotics, regular wound care and possible surgical intervention. Treatment completion is key to optimising outcomes, however the degree of and barriers to this are not well understood. Recent change from injectable treatment (SR8) to oral treatment (CR8) has made it feasible to further decentralise care, potentially improving treatment access and completion. However, the impact of this and of other demographic and clinical influences on treatment completion must be explored first to ensure appropriate models of care are developed. Methodology/Principal findingsA retrospective clinical notes review and secondary data analysis of records from patients diagnosed between 1 January 2006-31 December 2018 at four district hospital clinics in the Ashanti and Central Regions, Ghana. Univariable analyses and multivariable logistic regression were performed to assess the association between explanatory variables and treatment completion.There were 931 patient episodes across the four clinics with overall treatment completion of 84.4%. CR8 was associated with higher treatment completion compared to SR8 (OR 4.1, P = 0.001). There was no statistically significant association found between distance from patient residence to clinic and treatment completion. Conclusions/SignificanceImproved treatment completion with CR8 supports its use as first line therapy and may enable decentralisation to fully community-based care. We did not find an association between distance to care and treatment completion, though analyses were limited by data availability. However, we did find evidence that distance to care continues to be associated with more severe forms of disease, which may reflect the higher costs of accessing care PLOS NEGLECTED TROPICAL DISEASES
Background The declaration of COVID-19 as a pandemic on March 11 2020, by the World Health Organisation prompted the need for a sustained and a rapid international response. In a swift response, the Government of Ghana, in partnership with Zipline company, launched the use of Unmanned Automated Vehicles (UAV) to transport suspected samples from selected districts to two foremost testing centres in the country. Here, we present the experiences of employing this technology and its impact on the transport time to the second largest testing centre, the Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR) in Kumasi, Ghana. Methods Swab samples collected from suspected COVID-19 patients were transported to the Zipline office by health workers. Information on the samples were sent to laboratory personnel located at KCCR through a WhatsApp platform to get them ready to receive the suspected COVID-19 samples while Zipline repackaged samples and transported them via drone. Time of take-off was reported as well as time of drop-off. Results A total of 2537 COVID-19 suspected samples were received via drone transport from 10 districts between April 2020 to June 2021 in 440 deliveries. Ejura-Sekyedumase District Health Directorate delivered the highest number of samples (765; 30%). The farthest district to use the drone was Pru East, located 270 km away from KCCR in Kumasi and 173 km to the Zipline office in Mampong. Here, significantly, it took on the average 39 minutes for drones to deliver samples compared to 117 minutes spent in transporting samples by road (p<0.001). Conclusion The use of drones for sample transport during the COVID-19 pandemic significantly reduced the travel time taken for samples to be transported by road to the testing site. This has enhanced innovative measures to fight the pandemic using technology.
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