Browsing by Author "Horsfield, C."

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    Cytokine mRNA Expression in Mycobacteriam ulcerans-Infected Human Skin and Correlation with Local Inflammatory Response
    (American Society for Microbiology, 2006-05) Phillips, Richard Odame; Horsfield, C.; Mangan, J.; Laing, K.; Etuaful, S.; et.al
    Cytokine mRNA expression in biopsies of Mycobacterium ulcerans-infected human tissue was investigated using real-time PCR, and the findings were correlated with the clinical stages of disease and histopathologies. A broad range of cytokine mRNAs were detected in 16 early nodules and 28 late-stage ulcers, including those for the Th1 cytokines tumor necrosis factor alpha (TNF- ) and gamma interferon (IFN- ) and the Th2 cytokine interleukin 10 (IL-10). IFN- was strongly expressed in both nodules and ulcers, suggesting that a Th1 response begins early in the disease. There was a significantly higher expression of IL-8 and other proinflammatory cytokines in results from 32 biopsies with neutrophilia than in those from 12 biopsies without acute inflammation. Ten tissue samples containing granulomas showed high mRNA expression for IFN- , IL-1 , IL-12p35, IL-12p40, IL-15, and TNF- relative to 34 tissue samples without granulomas. These results suggest that the human immune response to M. ulcerans is similar to that seen with some other mycobacteria despite the presence of the toxin mycolactone in the tissues.
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    Cytokine Response to Antigen Stimulation of Whole Blood from Patients with Mycobacterium ulcerans Disease Compared to That from Patients with Tuberculosis
    (American Society for Microbiology, 2006-02) Phillips, Richard Odame; Horsfield, C.; Kuijper, S.; Sarfo, Fred Stephen; Obeng-Baah, J.; et.al
    Mycobacterium ulcerans disease (Buruli ulcer) is a skin-ulcerating infection common in some parts of the tropics. We have investigated cytokine secretion after stimulation of whole blood from Buruli ulcer (BU) patients in a region of endemicity in Ghana with M. ulcerans sonicate or culture filtrate antigens to investigate the development of the response over time and its specificity by comparison with the response to Mycobacterium tuberculosis sonicate in human immunodeficiency virus-negative tuberculosis patients. Significant gamma interferon (IFN- ) production in response to whole-blood stimulation with M. ulcerans sonicate was detected in patients with ulcers, which was higher than that in patients with nodules but similar to subjects with healed BU. The mean IFN- response in household contacts of BU patients was not significantly different from that in healthy control subjects from an area of nonendemicity. Results in patients with untreated, smear-positive pulmonary tuberculosis and tuberculosis patients on treatment for more than 2 weeks showed that BU patients responded better to M. ulcerans antigens than tuberculosis patients. In contrast, interleukin-10 results were higher in patients with active M. ulcerans disease than in those with healed lesions, but the pattern of response was similar to that seen in tuberculosis. A similar pattern of cytokine secretion was found using M. tuberculosis sonicate as an antigen. Neither of the two culture filtrate antigens of M. ulcerans appeared to be more specific than M. ulcerans sonicate. In the early stages of M. ulcerans disease there was a mixed Th1 and Th2 cytokine response, but the Th1 response emerged as the dominant type.
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    Sensitivity of PCR Targeting the IS2404 Insertion Sequence of Mycobacterium ulcerans in an Assay Using Punch Biopsy Specimens for Diagnosis of Buruli Ulcer
    (American Society for Microbiology, 2015-08) Phillips, Richard Odame; Horsfield, C.; Kuijper, S.; Lartey, A.; Tetteh, I.; et. al
    Punch biopsy specimens from Mycobacterium ulcerans disease lesions were used to compare the sensitivities and specificities of direct smear, culture, PCR, and histopathology in making a diagnosis of M. ulcerans disease in a field setting. PCR for the insertion element IS2404 was modified to include uracil-N-glycosylase and deoxyuridine triphosphate instead of deoxythymidine triphosphate to reduce the risk of cross contamination. The “gold standard” for confirmation of clinically diagnosed Buruli ulcer was a definite histological diagnosis, a positive culture for M. ulcerans, or a smear positive for acid-fast bacilli (AFB), together with a possible histological diagnosis. For 70 clinically diagnosed cases of M. ulcerans disease, the modified PCR was 98% sensitive and gave a rapid result. The sensitivities of microscopy, culture, and histology were 42%, 49%, and 82%, respectively. The use of a 4-mm punch biopsy specimen was preferred to a 6-mm punch biopsy specimen since the wound was less likely to bleed and to need stitching. Given adequate technical expertise and the use of controls, the PCR was viable in a teaching hospital setting in Ghana; and in routine practice, we would recommend the use of Ziehl-Neelsen staining of biopsy specimens to detect AFB, followed by PCR, in AFBnegative cases only, in order to minimize costs. Histology and culture remain important as quality control tests, particularly in studies of treatment efficacy.