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Item Molecular Characterization Of Previously Non-Typeable Rotavirus Strains In Ghana(University of Ghana, 2015-10) Damanka, S.A.Background Rotavirus is associated with severe infantile diarrhoea requiring hospitalization. In order to control severe disease caused by rotavirus infection, vaccination is considered an essential strategy. Currently, two rotavirus vaccines (Rotarix™ and RotaTeq™) have been included in the Expanded Program on Immunization of most African countries to help reduce the high disease indections. Ghana has over the past 20 years been involved in the surveillance of rotavirus diseases by isolating rotaviruses from diarrhoeic stools and determining the strain types as part of the build up to vaccine introduction. During rotavirus burden of disease surveillance between 2007 and 2011, three hundred and one rotavirus samples could not genotype with the pool of primers recommended for use within the WHO Regional Reference Laboratory network. Samples with sufficient stools were characterized. Aim The study sought to characterize and identify primer mismatches for nontypeable rotavirus strains at the Regional Rotavirus Reference laboratory in Ghana. Methodology One hundred and eleven G and 89 P-types had sufficient stool and were characterized by sequencing methods. To confirm previous EIA results, 10% of randomly selected nontypeable samples were reanalyzed using the commercially available DAKO IDEIA kit (Dako Diagnostics, Cambridgeshire, UK). To assess the integrity of RV dsRNA, Polyacrylamide Gel Electrophoresis (PAGE) was carried out. Viral ribonucleic acids were extracted by the phenol-chloroform-guanidine-isothiocyanate method and one step RT-PCR targeting the VP7 and VP4 genes of RVA was carried out using PCR primer pairs, Beg9/End9 and 9Con1/9Con2 for VP7 xv University of Ghana http://ugspace.ug.edu.gh genes and Con2/Con3 and VP4F/VP4R for VP4 genes. The RT-PCR products were purified with the QIAquick PCR purification kit (Qiagen/Westburg), sequenced with the ABI PRISM® BigDye Terminator v.3.1 Ready Reaction mix. Amplicons were purified using ethanol-sodium acetate precipitation method. Sequences were subjected to BLAST (Basic Local Alignment Search Tool) and subsequently characterized using the web-based genotyping tool, RotaC v2.0. Results The G-types were determined for 74 out of the 111 samples that had sufficient stool to work with. The G-types detected were as follows: G1; 31 (41.8%), G2; 9 (12.1%), G3; 11 (14.8%), G6; 1 (1.4%), G8; 2 (2.7%), G9; 14 (18.9%) and G12; 6 (8.1%). Sequence analyses of the Ghanaian VP7 isolates and cognate genes of known ancestral and contemporary reference strains revealed the accumulation of point mutations in the antigenic regions. The P-types were successfully characterized for 57 of the 89 samples that had sufficient stool. Forty-eight (84.2%) of these were identified as P[8]a strains of which 5 (10.4%) were characterized as the rare OP354-like human rotavirus P[8]b subtype. Other strains detected in the study were P[4], 1 (1.8%), P[6], 7 (12.3%) and P[14], 1 (1.8%). P[14] was found in combination with G6. In Silico PCR performed with in-house primers failed to genotype Ghanaian P[8] isolates. However, In Silico PCR with published primers, Rev compl 1T-1D and P8b-MMC38 successfully genotyped the Ghanaian P[8]a and P[8]b isolates respectively. Discussion, conclusion and recommendation The study determined the identity of previously nontypeable rotavirus strains in Ghana and established their genetic relatedness to globally circulating strains. The study also showed that the VP7 and VP4 genes of Ghanaian rotavirus strains have evolved as a result of accumulated point mutations over the years which might have led to genotyping failure. The amino acid xvi University of Ghana http://ugspace.ug.edu.gh differences detected in the antigenic regions of the Ghanaian isolates may lead to conformational changes that may influence the effectiveness of the vaccine. The study revealed for the first time the detection of OP354-like ([P[8]b) genotype in Ghana. It also identified for the first time G6P[14] rotavirus strain in Ghana. The study highlights the importance of including sequencing in the characterization of rotavirus strains as well as regularly updating the primer sequences employed for molecular typing of rotaviruses. Continuous surveillance of circulating rotavirus strains is important for the detection of new rotavirus strains as well as for the evaluation of the rotavirus vaccination program in Ghana.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.Item Detection and Characterization of Human Parainfluenza Viruses in Ghanaian Children Below Five Years with Acute Lower Respiratory Tract Infections(University of Ghana, 2014-06) Fiave, C. A.; Adiku, T. K.; Ampofo, W. K.; University of Ghana, College of Health Sciences , School of Biomedical and Allied Health Sciences , Department of Medical MicrobiologyWorldwide, acute lower respiratory tract infections in young children are one of the leading causes of childhood mortality. The need to investigate respiratory viruses associated with acute lower respiratory tract infections, more especially all four types of human parainfluenza viruses in Ghana has become crucial due to the limited information available on these viruses. Thus the aim of this study was to detect and characterize human parainfluenza viruses in children that present with acute lower respiratory tract infections at Princess Marie Louise Children’s Hospital in Accra, Ghana. From March to July 2013, nasopharyngeal swabs were collected from 71 children and the presence of human parainfluenza viruses was investigated. Ribonucleic acid extracts of the nasopharyngeal swabs were subjected to one-step real time reverse transcriptase polymerase chain reaction. Positive samples were further analysed molecularly. Also, the demographics of participants, risk factors for human parainfluenza viruses infection as well as clinical presentations were also evaluated. A 19.7% positive test rate was recorded for the human parainfluenza viruses. All four types of the virus were detected but with different frequencies (five were type 1, one was type 2, six were type 3 and two were type 4). No multiple serotype infection of human parainfluenza viruses was detected in the positive cases. Positivity was highest (57.1%) in children who were less than one year old. Males and females were equally infected. Phylogenetic analysis of the human parainfluenza viruses sequenced revealed that the strains within each type were closely related and clustered with reference strains. Age and having siblings with respiratory tract infections were risk factors significantly associated with the human parainfluenza viruses’ infection. In conclusion, all four types of human parainfluenza viruses are in circulation in Ghana and were associated with acute lower respiratory tract infections.Item Hiv-1 Drug Resistance Profiles of Ghanaian Women on Antiretroviral Therapy After Prevention of Mother-To-Child Transmission (Pmtct)(University of Ghana, 2014-07) Martin-Odoom, A.; Adiku, T.K.; Ampofo, W.K.; Lartey, M.; Blanco, E.D.; University of Ghana, College of Health Sciences , School of Biomedical and Allied Health Sciences , Department of Medical MicrobiologyFrom 2007 to 2011 HIV-positive pregnant women in Ghana, were given a combination of Zidovudine and Lamivudine as prophylaxis during pregnancy and a single dose of Nevirapine upon the onset of labour towards the prevention of mother to child transmission (PMTCT) of HIV. Since these women were subsequently given the same antiretrovirals (ARVs) to manage their HIV infection, there was therefore the need to determine the HIV-1 drug resistance profiles in such patients in Ghana. This study sought to investigate HIV-1 drug resistance associated mutations present in mothers on ART who had previously received ART prophylaxis for PMTCT in selected centres in Ghana. Genotypic Drug Resistance Testing for HIV-1 was carried out on study participants to identify any HIV-1 drug resistance associated mutations present and HIV viral load and CD4 counts were also measured. There were three categories of HIV-1 patients encountered in this study- Concordant Responders (had increased CD4 with viral suppression to undetectable levels), Immunological Responders (had increased CD4 but with detectable viremia) and Immunological Non-Responders (had decreased CD4 with viral suppression to undetectable levels). Participants who had prophylaxis before HAART, those who had HAART without prophylaxis and the drug-naïve participants had 32% (8), 5% (3) and 15% (4) HIV-1 drug resistance associated mutations (DRAMs) respectively. Thirty-five percent (35%) had nucleoside reverse transcriptase inhibitor (NRTI) and non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance associated mutations(RAMs) in the RT gene and 1 (3%) had RAMs in the PR gene for PIs. The most common NRTI mutation found was M184V; K103N and A98G were the most common NNRTI mutation found in this study. Thymidine Analogue Mutations (TAMs) such as M41L, D67N, K70R, L210W, T215Y/F and K219E were found in all the groups; the most common of the TAMs found in the study is M41L and T215Y. The major resistance mutation to Protease Inhibitors (PIs) seen in the study was I84V. Two HIV-1 drug-naïve patients in the Eastern region had mutations that conferred resistance to both nucleoside reverse transcriptase inhibitors (M184V and L210W) and non-nucleoside reverse transcriptase inhibitors (K103N and V106A). For the Reverse Transcriptase gene, 33(83%) of samples were of subtypes CRF02_AG, 2(5%) were subtype CRF01_AE, 1(3%) was subtype A, 2 (5%) were subtype B and 2 (5%) were subtype G. With the Protease gene, 32 samples (97%) were subtype CRF02_AG and 1(3%) was subtype A. HIV-1 positive Ghanaian mothers who had prophylaxis prior to initiating ART had immunological, virological and HIV-1 drug resistance associated mutations profiles that have adverse effect on their clinical profiles, resulting in sub-optimal responses to ART. Emergence of transmitted HIV-1 drug resistance could be on the increase and needs to be monitored. Drug resistance testing and early initiation of treatment upon diagnosis would help to produce a better treatment outcome for Ghanaian HIV-1 positive mothers and pregnant women. The study has provided useful baseline information on the profiles of Ghanaian women on ART after prophylaxis.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 Epidemiology and Molecular Characterization of Giardia Lamblia and Cryptosporidium Sp. Infections among Children In Accra, Ghana(University of Ghana, 2013-07) Anim-Baidoo, I.; Ayeh-Kumi, P.F.; Gyan, B.; Adjei, A.A.Giardiasis and cryptosporidiosis remain as part of the commonest gastroenteritis in Ghana. The diseases are caused by the protozoan parasites, Giardia lamblia and Cryptosporidium sp. respectively. Inadequate supply of treated water and poor sanitation are some of the key factors leading to the spread of these infections. Being zoonotic diseases, it is suspected that, a large proportion of human infections could come from infected domestic and farm animals. Though, use of molecular tools has helped to understand how the diseases spread in humans, animals, and the environment, very little information is available on the epidemiology and transmission routes of G. lamblia and Cryptosporidium sp. in Ghana, and a genetic characterization of the parasite has also not been thoroughly investigated. Information on clinical manifestations of Giardia infections and co-infections with other diarrhoeal causing agents particularly rotavirus remain scanty. In the present study, the epidemiology and molecular characterization of the two parasitic infections were investigated. The study, a prospective cross-sectional hospital and community-based, was conducted in Accra, Ghana. A total of 485 patients comprising of 365 diarrhoeic and 120 non-diarrhoeic children of age ≤ 5 years, were studied. Stool samples were tested microscopically, and by enzyme immunoassay kits. Positive samples were tested by the semi- nested polymerase chain reaction (PCR) and subsequently characterized into genotypes by PCR-RFLP, and nucleotide sequence analysis. Demographic and clinical data were obtained by a structured questionnaire. In the hospital-based study, prevalence rates of 5.8% and 22.0% were observed for G. lamblia and Cryptosporidium sp. infections respectively, and prevalence in diarrhoeic children was significantly higher than non-diarrhoeic children (P< 0.0001). Infection in day care centres was 10.1% for G. lamblia and 4.2% for Cryptosporidium. Neither gender nor breastfeeding habits, education level of mother, presence of domestic animals, source of children’s food, seasons (dry or rainy) was a risk factor for infections of the two parasites. However, age and source of drinking water were identified as associated risk factors for infection. G. lamblia genotype B and Cryptosporidium parvum were identified in the genotyping study. Although severity of rotaviral diarrhoea was reduced by Giardia co-infection, the results cannot be conclusive. Although both parasites were present in the studied population, cryptosporidial diarrhoea appears to be more common than giardial diarrhoea. The presence of infections among non-diarrhoeal children is of much concern, as they can spread infections unknowingly. The presence of genotype B as the only prevailing genotype of G. lamblia indicates that infections from animals will be uncommon, but Cryptosporidium parvum transmission could be either anthroponotic or zoonotic. The co- infection study had a limitatio n, and therefore demands further investigation. Several Cryptosporidium isolates that were successfully sequenced but whose identity were not clear, need further investigation as they could be new species emerging.