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Browsing by Author "Frimpong, Michael"

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Cellular immune response to mycobacterium ulcerans infection
(2015-07-12) Frimpong, Michael
Background: M. ulcerans infection (Buruli ulcer) is a disease of the skin and soft tissue endemic in sub-Sahara Africa, with the major burden in West Africa. It belongs to a large group of environmental mycobacteria. Generally, protection against mycobacterial infection is thought to be based on cell-mediated immunity, specifically Th-1 type cellular immune responses are essential for control of mycobacterial infections, while humoral response have little benefit. The reasons why only some individuals in endemic areas who are exposed to M. ulcerans develop lesions are not known but are likely to reflect individual differences in the immune response to infections with this mycobacterium. Aim of study: These study aims at describing the cellular immune response to Mycobacterium ulcerans infection (Buruli ulcer) associated with protection and determine the efficacy of two subunit proteins (MUL 4978 and MUL 3720) as potential candidates for vaccine against Buruli ulcer. And finally investigate the effect of co-infection with M. perstans on BU susceptibility. Methods: All clinically suspected cases of Buruli ulcer were confirmed by standard PCR and ZN microscopy. Interferon gamma (IFN-γ) secretion following stimulation with mycobacterial antigens of peripheral blood mononuclear cells (PBMC) and whole blood from Buruli ulcer confirmed patients and household contacts were investigated using IFN-γ ELISA. The effectiveness of two subunit protein vaccines (MUL 3720 and MUL 4978) as potential vaccine candidates was tested using the method mention above. Also CD4+, CD8+ T-cell profiles and CD19 + cell populations in patients with Buruli ulcer were determined by flow cytometry analysis. A case control of 66 M. ulcerans (Mu) disease patients and 30 household contacts were investigated for Mansonella perstans (Mp) co-infection. Patients confirmed to have Mu disease vi by PCR were given the WHO recommended combination of rifampicin 10mg/kg and streptomycin 15mg/kg for 8 weeks. Ivermectin 150ug/kg and doxycycline 200mg were administered for 6 weeks as treatment of Mp infection when present. Results: The results showed that following stimulation with M. ulcerans antigens, PBMC from Buruli ulcer patients and their household contacts mounted high IFN-γ response. Also the Buruli ulcer patients with ulcerative lesions produced more IFN-γ than those with pre-ulcerative lesions (p = 0.026). IFN-γ secretion increased with treatment, with significant difference (p = 0.0078) at 6 weeks compared to baseline (pre-treatment), corresponding to a decrease in patients’ lesion sizes. Patients with severe forms of Buruli lesions (Category II and III) had a significantly decreased CD4+ T-cell population compared with healthy contacts (p = 0.0395). There were no statistically significant differences in the populations of CD8+ T-cell and B cell (CD3-CD19+) populations. There were high IFN-γ responses to subunit protein vaccines and IFN-γ levels in both candidates vaccine antigens (MUL 3720 and MUL 4978) were significantly high after 6 weeks of treatment compared to before treatment (p = 0.03 and 0.005) respectively. Fifteen out of 66 (23%) patients with Mu disease were co-infected with Mp while 4 out of 30 (13%) of the household contacts had infection with Mp. While filarial infection was more common among Buruli ulcer patients than household contacts it did not influence healing time of Buruli lesions (p = 0.93) or predispose patients to more severe forms of the disease. Conclusions: These findings suggest that T helper cell-1 (Th-1) immune response to M. ulcerans may play a protective role in the control of the disease. Also patients with severe forms of Buruli ulcer had depleted CD4+ T-lymphocyte populations. Furthermore, the results indicate that subunit protein vaccines MUL 3720 and MUL 4978 are immunogenic in human ex-vivo assays. This study also provided clear evidence of M. perstans co-infection in Buruli ulcer patients.
Clinical and microbiological predictors of healing in Buruli ulcer disease
(Journal of Clinical Tuberculosis and Other Mycobacterial Diseases, 2024-02) Agbavor, Bernadette; Agbanyo, Abigail; Laglo, Aloysius Dzigbordi; Antwi, Philemon Bosiako; Ackam, Nancy; Adjei, Jonathan; Frimpong, Venus; Boampong, Kwadwo; Frimpong, Michael; Addo, Matthew Glover; Wansbrough-Jones, Mark; Amoako, Yaw Ampem; Phillips, Richard Odame; 0000-0001-5014-6153
Introduction: Wound measurements are relevant in monitoring the rate of healing (RoH) and may predict time to healing. Predicting the time to healing can help improve the management of Buruli ulcer. We examine three methods for the determination of RoH and their use as predictors of time to healing. Methods: Lesion measurements of Buruli ulcer patients treated from 2007 to 2022 were obtained with acetate sheet tracings (2D) or Aranz software (3D) fortnightly. RoH was determined using the absolute area, percentage area reduction and linear methods at 4 weeks post onset of antibiotic treatment. Predicted time to healing was compared to the actual healing time. Baseline characteristics were assessed for associations with healing. Results: All three methods for calculating the RoH significantly distinguished between fast and slow healers (p <0.0001). The predicted healing time using the linear method was comparable to the actual healing time for fast healers (p = 0.34). The RoH was influenced by the form of lesion, with plaques [OR 2.19 5 %CI (1.2–3.6), p =0.009], and oedemas [OR 8.5; 95 %CI (1.9––36.9), p = 0.004] being associated with delayed healing. The proportion of patients with paradoxical reactions 16 % vs 3 %, p < 0.0001), higher baseline bacterial load (75/104;72 % vs 21/47;45 %, p = 0.001) and delayed clearance of viable organisms (71/104;68 % vs 9/47;19 %, p <0.0001) was higher in the slow healers than the fast healers. Conclusion: Predicted healing rates were comparatively lower for slow healers than fast healers. Baseline characteristics associated with healing can be explored for an improved disease management plan to reduce patient and caregiver anxiety.
Combined Inflammatory and Metabolic Defects Reflected by Reduced Serum Protein Levels in Patients with Buruli Ulcer Disease
(PLOS Neglected Tropical Diseases, 2014-04-10) Phillips, Richard Odame; Sarfo, Fred Stephen; Landier, Jordi; Oldenburg, Reid; Frimpong, Michael; et. al
Buruli ulcer is a skin disease caused by Mycobacterium ulcerans that is spreading in tropical countries, with major public health and economic implications in West Africa. Multi-analyte profiling of serum proteins in patients and endemic controls revealed that Buruli ulcer disease down-regulates the circulating levels of a large array of inflammatory mediators, without impacting on the leukocyte composition of peripheral blood. Notably, several proteins contributing to acute phase reaction, lipid metabolism, coagulation and tissue remodelling were also impacted. Their down-regulation was selective and persisted after the elimination of bacteria with antibiotic therapy. It involved proteins with various functions and origins, suggesting that M. ulcerans infection causes global and chronic defects in the host’s protein metabolism. Accordingly, patients had reduced levels of total serum proteins and blood urea, in the absence of signs of malnutrition, or functional failure of liver or kidney. Interestingly, slow healers had deeper metabolic and coagulation defects at the start of antibiotic therapy. In addition to providing novel insight into Buruli ulcer pathogenesis, our study therefore identifies a unique proteomic signature for this disease.
Diagnostics for COVID-19: A case for field-deployable, rapid molecular tests for community surveillance
(Ghana Med J., 2020) Frimpong, Michael; Amoako, Yaw A.; Anim, Kwadwo B.; Ahor, Hubert S.; Yeboah Richmond; Arthur, Joshua; Dakorah, Justin S.; Gborgblovor, Delphine; Akrofi, Samuel; Owusu, Michael; Sylverken, Augustina Angelina; Binger, Tabea; Phillips, Richard Odame; Djan, Phyllis Sekyi; 0000-0003-1901-6793; 0000-0002-4642-789X; 0000-0001-5066-150X
Across the globe, the outbreak of the COVID-19 pandemic is causing distress with governments doing everything in their power to contain the spread of the novel coronavirus (SARS-CoV-2) to prevent morbidity and mortality. Actions are being implemented to keep health care systems from being overstretched and to curb the outbreak. Any policy responses aimed at slowing down the spread of the virus and mitigating its immediate effects on health care systems require a firm basis of information about the absolute number of currently infected people, growth rates, and locations/hotspots of infections. The only way to obtain this base of information is by conducting numerous tests in a targeted way. Currently, in Ghana, there is a centralized testing approach, that takes 4-5 days for samples to be shipped and tested at central reference laboratories with results communicated to the district, regional and national stakeholders. This delay in diagnosis increases the risk of ongoing transmission in communities and vulnerable institutions. We have validated, evaluated and deployed an innovative diagnostic tool on a mobile laboratory platform to accelerate the COVID-19 testing. A preliminary result of 74 samples from COVID-19 suspected cases has a positivity rate of 12% with a turn-around time of fewer than 3 hours from sample taking to reporting of results, significantly reducing the waiting time from days to hours, enabling expedient response by the health system for contact tracing to reduce transmission and additionally improving case management.
IFN-g and IL-5 whole blood response directed against mycolactone polyketide synthase domains in patients with Mycobacterium ulcerans infection
(PeerJ, 2018-07-31) Phillips, Richard Odame; Loglo, Aloysius D.; Frimpong, Michael; Duah, Mabel Sarpong; Sarfo, Fred Stephen; et. al
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-g (IFN-g) 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-g 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-g 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-g and IL-5 responses of patients improved after treatment when compared to baseline results. Discussion: The major response to PKS antigen stimulation was IFN-g 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.
Infection with Mansonella perstans Nematodes in Buruli Ulcer Patients, Ghana
(Emerging Infectious Diseases, 2014-06-06) Phillips, Richard Odame; Frimpong, Michael; Sarfo, Fred Stephen; Kretschmer, Birte; Beissner, Marcus; et. al
During August 2010–December 2012, we conducted a study of patients in Ghana who had Buruli ulcer, caused by Mycobacterium ulcerans, and found that 23% were coinfected with Mansonella perstans nematodes; 13% of controls also had M. perstans infection. M. perstans co-infection should be considered in the diagnosis and treatment of Buruli ulcer.
Metabolomic profiles delineate mycolactone signature in Buruli ulcer disease
(Scientific Reports, 2015-12-04) Phillips, Richard Odame; Niang, Fatoumata; Sarfo, Fred Stephen; Frimpong, Michael; Guenin-Macé, Laure; et. al
Infection of human skin with Mycobacterium ulcerans, the causative agent of Buruli ulcer, is associated with the systemic diffusion of a bacterial macrolide named mycolactone. Patients with progressive disease show alterations in their serum proteome, likely reflecting the inhibition of secreted protein production by mycolactone at the cellular level. Here, we used semi-quantitative metabolomics to characterize metabolic perturbations in serum samples of infected individuals, and human cells exposed to mycolactone. Among the 430 metabolites profiled across 20 patients and 20 healthy endemic controls, there were significant differences in the serum levels of hexoses, steroid hormones, acylcarnitines, purine, heme, bile acids, riboflavin and lysolipids. In parallel, analysis of 292 metabolites in human T cells treated or not with mycolactone showed alterations in hexoses, lysolipids and purine catabolites. Together, these data demonstrate that M. ulcerans infection causes systemic perturbations in the serum metabolome that can be ascribed to mycolactone. Of particular importance to Buruli ulcer pathogenesis is that changes in blood sugar homeostasis in infected patients are mirrored by alterations in hexose metabolism in mycolactone-exposed cells.
Microscopy for Acid Fast Bacilli: A Useful but Neglected Tool in Routine Laboratory Diagnosis of Buruli Ulcer
(Journal of Tropical Diseases, 2015-05-18) Phillips, Richard Odame; Frimpong, Michael; Sarpong-Duah, Mabel; Beissner, M; Sarfo, Fred Stephen; et. al
Background: Laboratory diagnosis of Buruli ulcer disease has become vital with the introduction of antibiotic treatment. Polymerase chain reaction (PCR) for the IS2404 repeat sequence of Mycobacterium ulcerans is the gold standard for laboratory diagnosis. This is expensive and only carried out in reference laboratories in endemic countries in Africa. In order to improve the efficiency of diagnosis at the point of care and reduce the total cost of patient management, we decided to evaluate Ziehl-Neelsen (ZN) staining for acid-fast bacilli (AFB) as an inexpensive diagnostic tool. Methods: Two smears directly prepared at the point of care were examined under oil-immersion microscopy after ZN staining for AFB and compared the results to PCR samples from the same patients. Results: Good quality smears were obtained from all subjects and our results showed that when a second smear was added the sensitivity of microscopy for AFB was increased from 52 to 55% for FNA samples and from 51 to 57 % for swabs. Conclusion: If PCR were to be omitted in all patients with suspected Buruli ulcer disease when AFB were detected it would result in a considerable saving.
Paradoxical reactions in Buruli ulcer after initiation of antibiotic therapy: Relationship to bacterial load
(PLOS Neglected Tropical Diseases, 2019-08-26) Phillips, Richard Odame; Frimpong, Michael; Agbavor, Bernadette; Duah, Mabel Sarpong; Loglo, Aloysius; et. al
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.
Providing insight into the incubation period of Mycobacterium ulcerans disease: two case reports
(Journal of Medical Case Reports, 2019-07-18) Phillips, Richard Odame; Amoako, Y. A.; Frimpong, Michael; Awuah, D. O.; Plange-Rhule, G.; et. al
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.
Rapid detection of Mycobacterium ulcerans with isothermal recombinase polymerase amplification assay
(PLOS Neglected Tropical Diseases, 2019-02-01) Phillips, Richard Odame; Frimpong, Michael; Ahor, Hubert Senanu; Wahed, Ahmed Abd El; Agbavor, Bernadette; et. al
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.
Rapid Extraction Method of Mycobacterium ulcerans DNA from Clinical Samples of Suspected Buruli Ulcer Patients
(Diagnostics, 2019-11-26) Phillips, Richard Odame; Frimpong, Michael; Ahor, Hubert Senanu; Sakyi, Samuel Asamoah; Agbavor, Bernadette; et. al
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 e cient protocol for the extraction and detection of M. ulcerans DNA in clinical samples that is adaptable to field conditions.
Reply to “Compliance with Antimicrobial Therapy for Buruli Ulcer”
(Antimicrobial Agents and Chemotherapy, 2014-10) Phillips, Richard Odame; Sarfo, Fred Stephen; Abass, Mohammed K.; Frimpong, Michael; Ampadu, Edwin; et. al
Recently, Klis et al. conducted an audit of Buruli ulcer case record forms of patients managed under routine care conditions in a Buruli ulcer treatment center and showed a surprisingly high rate (54%) of noncompliance with therapy (1). Incomplete adherence to treatment has been identified as the most serious problem in tuberculosis control (2) and a major obstacle to the elimination of the disease (3). To ensure adherence to therapy in our study, several approaches were incorporated into patient care. These included issuing medication in 2-weekly batches, allowing the clinician several opportunities to assess adherence during therapy.
Simple, Rapid Mycobacterium ulcerans Disease Diagnosis from Clinical Samples by Fluorescence of Mycolactone on Thin Layer Chromatography
(PLOS Neglected Tropical Diseases, 2015-11-19) Wadagni, Anita; Frimpong, Michael; Phanzu, Delphin Mavinga; Phillips, Richard O.; Ablordey, Anthony
Mycobacterium ulcerans infection, known as Buruli ulcer, is a disease of the skin and subcutaneous tissues which is an important but neglected tropical disease with its major impact in rural parts of West and Central Africa where facilities for diagnosis and management are poorly developed. We evaluated fluorescent thin layer chromatography (f-TLC) for detection of mycolactone in the laboratory using samples from patients with Buruli ulcer and patients with similar lesions that gave a negative result on PCR for the IS2404 repeat sequence of M. ulcerans Methodology/Principal findings Mycolactone and DNA extracts from fine needle aspiration (FNA), swabs and biopsy specimen were used to determine the sensitivity and specificity of f-TLC when compared with PCR for the IS2404. For 71 IS2404 PCR positive and 28 PCR negative samples the sensitivity was 73.2% and specificity of 85.7%for f-TLC. The sensitivity was similar for swabs (73%), FNAs (75%) and biopsies (70%).

Browsing by Author "Frimpong, Michael"

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