Department of Medical Microbiology
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Browsing Department of Medical Microbiology by Author "Newman, M.J."
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Item Aetiology, Malnutrition and Faecal Lactoferrin Levels in Paediatric Diarrhoea(University of Ghana, 2011-08) Opintan, J.A.; Newman, M.J.; Wilson, M.D.; Guerrant, R.L.; University of Ghana, College of Health Sciences, School of Biomedical and Allied Health Sciences, Department of Medical MicrobiologyDiarrhoea is a major public health problem that affects the physical and cognitive development of young children. Anthropometric data was collected from 274 children, 170 with diarrhoea and 104 without diarrhoea. Stool specimens were analyzed by conventional culture, PCR for EAEC, Shigella, Cryptosporidium, Entamoeba, and Giardia species, as well as by ELISA for faecal lactoferrin levels. Additionally, all E. coli recovered from culture were PCR screened for EAEC, and compared with those obtained from the stool DNA. Multiple gene loci (aaiC, aap, attA and aggR) were sought for EAEC. About 50% of the study population was mildly to severely malnourished. Mild to severe malnutrition (WAZ <-l), moderate to severe stunting (HAZ < -2) and moderate to severe wasting (WHZ < -2) were associated with diarrhoea (p = 0.023, 0.026 and 0.062, respectively). In only 1 of 170 diarrhoea stool specimen was Shigella flexneri recovered by culture. EAEC and Cryptosporidium were associated with diarrhoea (p = 0.048 and 0.011, respectively), and malnourished children who had diarrhoea were often co-infected with both Cryptosporidium and EAEC. About 27 % (4/15) C. parvum genotypes were identified by HRM analysis. Faecal lactoferrin levels were higher in children with diarrhoea (p = 0.019). Children who had EAEC infection, with or without diarrhoea had high mean lactoferrin levels irrespective of nutritional status. In conclusion, the current study identified high levels of growth deficits among the children with/without diarrhoea. The use of DNAbiomarkers revealed that EAEC and Cryptosporidium were common intestinal pathogens in Accra, and that elevated faecal lactoferrin was associated with diarrhoea in this group of children. EAEC’s dispersin gene (aap) was significantly associated with diarrhoea in both the faecal and bacterial DNAs, in the children studied (p < 0.05). Publication: Part o f the data presented in this thesis is published as follows: Opintan JA et al. (2010). Pediatric Diarrhea in Southern Ghana: Etiology and Association with Intestinal Inflammation and Malnutrition. Am J. Trop Med Hyg. 83: 936 - 943.Item Phenotypic Characterization of Host-Pathogen Interaction on Mycobacterium Africanum(University of Ghana, 2015-12) Tetteh, J.K.A.; Newman, M.J.; Yeboah-Manu, D.; Gyan, B.; University of Ghana, College of Health Sciences School of Biomedical and Allied Health Sciences Department of Medical MicrobiologyBackground: Mycobacterium africanum (MAF) and Mycobacterium tuberculosis sensu strico (MTBss) are two members of a closely related bacterial species of Mycobacterium tuberculosis complex (MTBC) that causes tuberculosis (TB) in humans. However MAF is known to cause up to 50% of human pulmonary TB in West Africa only. MAF has been sub-divided into MAF West African 1 (MAF1) (Lineage 5) and MAF West African 2 (MAF2) (Lineage 6), as two distinct phylogenetical lineages within MTBC. Subsequently the absence of Mycobacterium tuberculosis deletion gene (TbD1) strains in MTBss has been referred to as modern lineage whilst ancient lineage (MAF1 and MAF2) have the presence of TbD1. Ghana represents one of the few countries within Central West Africa known to have this unique genetic diversity of MAF1, MAF2 and MTBss that causes TB cases in significant proportions. While previously it was thought MAF is genetically very closely related to MTBss such that there are no important phenotypic differences between the two species, current advance in molecular biology indicate that substantial genetic difference exit between the two that can translate into significant phenotypic differences including immunogenicity and virulence. Aim: The aim of the study was to analyze the phenotypic features of host-pathogen interaction in Mycobacterium africanum and compared to Mycobacterium tuberculosis sensu strico strains. Methodology: The study was embedded in 2 different projects. Retrospective archived cryopreserved peripheral blood mononuclear cells (PBMCs) of MAF-infected and MTBss-infected patients were stimulated with growth medium (negative control), Staphylococcus enterotoxin B (SEB, positive control) and recombinant early secreted antigenic protein 6 kiloDalton/culture filtrate protein 10 kiloDalton fusion protein (rESAT-6/CFP-10), surface stained for T-subsets (CD4 and CD8) and intracellular cytokine, interferon gamma (IFN-), and acquired with FACS Calibur flow cytometer. The second study used characterized large sequence polymorphism (LSP) clinical isolates identified as MAF1, MAF2 and MTBss to determine intracellular growth assay in human monocyte–derived macrophages (MDM), mean doubling time and pro-inflammatory tumour necrosis factor–alpha (TNF-α), interleukin 6 (IL-6) and 12p70 cytokines by enzyme-linked immunosorbent assay (ELISA). Results: The percentage frequencies of CD4+IFN-+ and CD8+ IFN-+ T cells of MAF-infected patients did not differ from the percentage frequencies CD4+ IFN-+ and CD8+ IFN-+ T cells of MTB-infected patients in response to rESAT-6/CFP-10 fusion protein (p>0.05). Uptake of MAF1, MAF2 and MTBss representing modern and ancient strains respectively at 4hours was not significant (p>0.05). Mean intracellular growth index from 24hours to 72hours was significantly rapid for MTBss (modern) lineage compared to MAF1 and MAF2 (ancient) lineages (p<0.05). In contrast the mean doubling time of MTBss (modern) lineage was significantly lower compared to MAF1 and MAF2 (ancient) lineages (p<0.05). Levels of pro-inflammatory cytokines released into the supernatants by MTBss, MAF1 and MAF2 at 4hours was not statistically significant (p>0.05). However at 24hours to 72hours levels released by MAF1 and MAF2 (ancient) lineages was significantly higher than MTBss (modern) lineage (p<0.05). Conclusion: The study has shown that MAF-infected patients had similar T subset response to rESAT-6/CFP-10 fusion protein relative to MTBss-infected patients. Furthermore MAF had reduced uptake, low intracellular growth rate and a higher doubling time in MDM. Likewise MAF (ancient) lineages have hyper-inflammatory response thereby inducing a ‘slow growth’ phenotype highlighting the point that MAF indeed has lower virulence and longer latency leading to slower progression to active disease in the host.