Browsing by Author "Agbavor, Bernadette"

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    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.
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    Comparative detection of Mycobacterium Ulcerans dna by PCR from FTA cards, cell Lysis solution and RNA protect solution
    (OCTOBER, 2016.) Agbavor, Bernadette
    Buruli ulcer is a necrotizing skin disease caused by Mycobacterium ulcerans. It occurs in more than 33 countries with tropical and subtropical conditions. Samples for diagnosis and confirmation are usually transported in liquid media including cell lysis solution (CLS), PANTA, and RNA protect solution from the disease centers to reference laboratories. FTA cards have been developed to store, preserve and protect nucleic acids stored on it. It serves as a transport medium for samples requiring the use of nucleic acids and reduces storage space. Diagnosis of Buruli ulcer requires transporting samples from the remote communities to the laboratories as well as for storage and sample collection. The usage of FTA cards has however not been explored in the field of Buruli ulcer and in Ghana. This study therefore sought to evaluate the use of FTA for obtaining DNA samples and transporting of M. ulcerans to the laboratories and to also establish its detection limit. Methods: Known concentrations of M. ulcerans suspensions were spotted on the FTA cards and allowed to air dry. Two millimeters of discs were cut out and DNA was extracted using the in-house extraction procedure to optimize the cards and determine the detection limit of the cards. Swab samples from ulcerative lesions and FNA samples from pre-ulcerative lesions were taken from 53 suspected Buruli ulcer patients and distributed into CLS, RNA protect solutions and spotted on the FTA cards. DNA was extracted from the samples and amplified using the Dry Reagent Based PCR for the CLS and FTA card samples and qPCR for RNA protect solution samples. Amplicons were viewed on agarose gel and results recorded as positive, negative or inhibited. Samples were also smeared on glass slides for the Zeihl Neelson stain for acid fast bacilli. Samples stored on the FTA cards were kept over a period of six months and extraction done on monthly basis to detect the preservative ability of the card. Comparisons were made and results analyzed using GraphPad prism version 5.0 and Stata version12.0. From the optimization test, it was observed that in absolute numbers, 1 bacterium in a concentration of 10 bacteria/ml spotted on the card can be detected. The sensitivity of the FTA cards using patient samples was recorded as 58% and a specificity of 70% whiles that of qPCR was 94% sensitivity and 67% for specificity when compared with the gold standard, the DRB PCR. When compared with the smear for microscopy, the sensitivity for FTA cards was 80% and a specificity of 48%. Samples stored over a period of two months had the highest positivity ratio of 100% followed by those stored for five months with 58.8% and 55%, 50%, 44%, and 27% for four months, one month, three months and six months respectively. inter-assay agreement between FTA PCR and standard CLS PCR was 50% and a p-value of 0.58 for swab samples and 67.7%, p value of 0.22 for FNA samples. FTA cards serve as a good storage and transport medium However because of low sensitivity and low inter-assay agreement rate, it cannot be used as a replacement for the CLS. It may however be used in addition to the smear for microscopy in areas where access to the reference laboratories is difficult.
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    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.
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    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.
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    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.