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Entamoeba histolytica is an invasive intestinal amoeba morphologically indistinguishable from Entamoeba dispar, a closely related organism that is not able to invade tissues. Differential diagnosis under conventional microscopy is therefore impossible. Reliable tools are needed for clinical diagnosis and for the reevaluation of the prevalence of infection with the invasive species worldwide. Monoclonal Antibody (MAb) 20/7D exhibited promising results when ascites was used to identify cultured isolates of E. histolytica by indirect immunofluorescence assays (IFA), and when used in a Faecal Antigen Capture Enzyme-Linked Immunosorbent Assay (FAC-ELISA) for laboratory diagnosis of amoebic dysentery and colitis. Here, further development of the assay was attempted to increase its sensitivity and use it for detection of asymptomatic carriers of E. histolytica. After purification and subsequent titration in ELISA, MAb 20/7D did not adequately distinguish between crude lysates of cultured E. histolytica and E. dispar trophozoites. MAb 20/7D reacted with a similar soluble antigen of E. histolytica and E. dispar, which confirmed previous observations in western blot analysis under non-reducing conditions. Therefore, the use of the FAC-ELISA for diagnosis in areas where E. dispar is endemic is probably not viable. A nucleic acid detection method was therefore developed. Polymerase Chain Reaction was used to amplify specific tandem sequences in the 24.5 Kb episome of E. histolytica and E. dispar. After PCR, internal sequences of digoxigenin-labelled PCR products were hybridized to specific biotin-labelled probes for E. histolytica or E. dispar and detected in Enzyme- Linked Immunosorbent Assay (ELISA). The Polymerase Chain Reaction Solution- Hybridisation Immunosorbent Assay (PCR-SHELA) was evaluated on samples from travellers returning from the tropics to Barcelona. The sensitivity and specificity were 98% and 100% respectively, when results were compared with microscopy. PCR-SHELA was also useful for differential diagnosis in cases of amoebic abscesses, amoebic dysentery, salmonellosis, ulcerative colitis and in asymptomatic carriage of E. histolytica. The new test gives sensitive and specific differentiation between E. histolytica and E. dispar in clinical specimens and it has proved successful in screening faecal samples in endemic areas for epidemiological purposes.

MSc (Med), Molecular Medicine and Haematology, Faculty of Health Sciences, University of the Witwatersrand, 2009 / This study assessed the effect that certain logistical and methodological factors in the laboratory could have on influencing precision and accuracy of enumeration of CD4+ cells. The efficacy of a new blood stabiliser to extend the window of CD4 testing, was also evaluated. CD4+ counts were derived using the 2-colour Pan-leucogating, 4-colour TetraONE and MultiTEST/TruCount protocols on the EPICS-XL, FC-500 or FACSCalibur flow cytometers. Statistical analyses included the paired-t-test, Spearman’s correlation and Bland Altman comparisons. The results showed that the reliability of CD4+ count results was heavily dependent on how blood samples were handled prior to and after receipt into the laboratory and on how samples were processed and analysed. The factors, motion, operator pipetting and analysis skills, storage temperature, use of different protocols, different gating strategies and the use of different flow cytometers, were found to influence accurate and precise enumeration of CD4+ counts.

We consider a Bayesian approach to the study of independence in a two-way contingency table obtained from a two-stage cluster sampling design. We study the association between two categorical variables when (a) there are no covariates and (b) there are covariates at both unit and cluster levels. Our main idea for the Bayesian test of independence is to convert the cluster sample into an equivalent simple random sample which provides a surrogate of the original sample. Then, this surrogate sample is used to compute the Bayes factor to make an inference about independence. For the test of independence without covariates, the Rao-Scott corrections to the standard chi-squared (or likelihood ratio) statistic were developed. They are ``large sample' methods and provide appropriate inference when there are large cell counts. However, they are less successful when there are small cell counts. We have developed the methodology to overcome the limitations of Rao-Scott correction. We have used a hierarchical Bayesian model to convert the observed cluster samples to simple random samples. This provides the surrogate samples which can be used to derive the distribution of the Bayes factor to make an inference about independence. We have used a sampling-based method to fit the model. For the test of independence with covariates, we first convert the cluster sample with covariates to a cluster sample without covariates. We use multinomial logistic regression model with random effects to accommodate the cluster effects. Our idea is to fit the cluster samples to the random effect models and predict the new samples by adjusting with the covariates. This provides the cluster sample without covariates. We then use a hierarchical Bayesian model to convert this cluster sample to a simple random sample which allows us to calculate the Bayes factor to make an inference about independence. We use Markov chain Monte Carlo methods to fit our models. We apply our first method to the Third International Mathematics and Science Study (1995) for third grade U.S. students in which we study the association between the mathematics test scores and the communities the students come from, and science test scores and the communities the students come from. We also provide a simulation study which establishes our methodology as a viable alternative to the Rao-Scott approximations for relatively small two-stage cluster samples. We apply our second method to the data from the Trend in International Mathematics and Science Study (2007) for fourth grade U.S. students to assess the association between the mathematics and science scores represented as categorical variables and also provide the simulation study. The result shows that if there is strong association between two categorical variables, there is no difference between the significance of the test in using the model (a) with covariates and (b) without covariates. However, in simulation studies, there is a noticeable difference in the significance of the test between the two models when there are borderline cases (i.e., situations where there is marginal significance).

This thesis carefully investigates some of the limitations of existing methods for atomic spectroscopic determination of mercury, selenium and arsenic in biological and environmental materials. In particular the need for adequate sample preparation to ensure reliable atomic spectroscopic determination of these metalloids was demonstrated extensively. The thesis evaluates four most commonly used wet digestion methods for the accurate determination of mercury in biological and environmental materials by cold vapour atomic absorption spectroscopy. Excellent recovery efficiencies were obtained with this digestion mixture in fish homogenate, horse kidney, soil, canned fish and hair samples for inorganic and organic mercury. The suitability of the digestion method for the reliable determination of mercury in soil, hair and canned fish samples was also demonstrated. Mercury levels in these samples were found within normal acceptable range. The thesis outlines a new simple procedure for overcoming the loss of mercury due to sample charring. It also described a new sample ultrasound low temperature wet digestion method for biological and environmental materials. The main advantage of this approach over other conventional methods is its ability to release all mercury in inorganic form from biological and environmental samples at much lower temperatures than reported earlier / Doctor of Philosophy (PhD)

When astronauts travel in space, their primary health hazards are high-energy cosmic radiations from galactic cosmic rays (GCR). Most galactic cosmic rays have energies between 100 MeV and 10 GeV. For occupants inside of a space shuttle, the structural material is efficient to absorb most of the cosmic-ray energy and reduce the interior dose rate to below 1.2 mGy per day. However, the biological effects of prolonged exposure to low-dose radiation are not well understood. The purpose of this research was to examine the feasibility of constructing a low-dose irradiation facility to simulate the uniform radiation field that exists in space. In this research, we used a pre-manufactured incubator, specifically the Thermo Scientific Forma Series II Water Jacketed CO2 Incubator, to act as shielding and simulate the exterior of the space shuttle. To achieve the desired dose rate (< 1 mGy/h) inside the incubator volume, the computer code MCNPX was used to determine required source activity and distance between the shielding and source. Once the activity and distance were calculated, an experiment was carried out to confirm the simulation results. The confirmation used survey meters and thermoluminescence dosimeters (TLDs) to map the radiation field within the incubator.

In a time of fierce global competition, where an immense amount of commercial messages reaches consumers around the clock and in every conceivable context, companies constantly seek to find new ways to reach out with their product. One way to do so, that has increased in popularity in recent years, is the use of gift bags. A gift bag may contain samples, miniature products and even products in full size, as a rule from several different brands, which the recipient receives free of charge. The gift bag is typically distributed at some form of event, such as store openings, fashion shows, theme parties and the like, and the receiver of the gift bag can then evaluate the content without any obligation to purchase. The ambition of the companies that choose to participate with their products in a gift bag is, of course, that the recipient will find their products satisfactory, and ideally, that he or she will continue to consume the brand in the future. There are numerous scientific research contributions related to the use of free samples, but very few that concern the gift bag, where a collection of samples from different sources and brands appear. The ambition of this study is to investigate how the conditions change when a free sample is exposed to competition from samples from other companies. The study has a qualitative approach, and consists of interviews with companies that use, or have used, gift bags as a marketing channel, along with an interview with a focus group consisting of people with experience of participating in various events where gift bags occur. The study concludes that marketing through the use of gift bags can have several positive effects for the participating brands. These are, for example, increased brand awareness and an increase in sales. However, it is of importance to evaluate the competing brands participating in the gift bag, as well as the context in which it will be distributed. The study shows that the size of the sample is of importance, since the receiver tends to notice more voluminous samples over smaller ones. Also, the presence of more established brands can have a positive effect on less well-known brands, as their credit will enhance the other participating products. It is important to note that this is only true for samples that are not of the same product category. If this is the case, they will instead be in direct competition with each other.

Human microbial research has become increasingly popular in biomedical areas due to the importance of role of human microbiome in human health. One purpose of studying human microbiome is to detect differentially abundant features from a limited group of subjects across biological conditions. Metagenomic analyses of the human microbial communities are extensively used for biomedical applications due to its reliable and evident comparative discoveries across more than one metagenomes when multiple communities are taken into consideration. Next-generation sequencing technology helps to detect taxonomic compositions of specific features/species contained in human microbial communities. Statistical analysis often starts by generating the Operational Taxonomic Units (OTUs) using taxonomic compositions to classify groups of closely associated human microbiomes. Oftentimes, the counts of features are observed as matched count data with excess zeros. Such data lead some differential abundance analysis methods to apply Zero-Inflated Poisson (ZIP) or Zero-Inflated Negative Binomial (ZINB) regression for modeling the microbial abundance. However, over-dispersion as well as within-subject variation and correlation of matched count data render the standard ZIP and ZINB regression inadequate. To account for the inherent within-subject variation and correlation, independent random effect terms are commonly included in the regressions. Therefore, a robust method that accounts the effect of matched samples and correlated random effects while considering over-dispersion and excess zeros of count data is need for statistical analysis. In this paper, a statistical method, the two-part correlated ZINB model with correlated random effects (cZINB), is proposed for testing the matched samples with repeated measurements.

Human adenoviruses (HAdV) are non-enveloped viruses with an icosahedral capsid and a linear double-stranded DNA genome. These viruses belong to the family Adenoviridae and genus Mastadenovirus. An important property of the HAdV is that it is non-enveloped making it highly resistant to detergents and harsh environmental conditions. This virus is grouped in seven species (A-G) with more than 88 genotypes. These seven species are associated with several diseases, such as, respiratory infections, keratoconjunctivitis, urinary infections, hepatitis and gastrointestinal infections. The HAdV is one of the etiological causes of acute gastroenteritis, mainly caused by HAdV-F40 and HAdV-F41. The virus can be transmitted via the faecal-oral route, inhalation of respiratory droplets and direct contact with contaminated environments. The virus is known to be ubiquitous in environments where human contamination is likely to occur such as wastewater treatment plants. These human contaminations could occur through contaminated secretion and excretions within aqueous environments. There is currently a limited amount of information on the HAdV in water Molecular characterisation of human adenoviruses from environmental environments, particularly in Tshwane, Gauteng. Therefore, the aim of the study was to investigate the presence and genotypes of human adenovirus in environmental samples namely raw sewage and treated effluent, using molecular methods. For genotypic characterisation, Sanger sequencing was used on amplicons from 12 HAdV positive samples and next generation sequencing were used on all the amplicons from HAdV positive samples. A total of 150 environmental samples (75 raw sewage and 75 effluent) were collected from two wastewater treatment plants in Tshwane over the study period of 18 months. These environmental samples comprised of 1 L raw sewage and 10 L treated effluent samples. The primary viral recovery for the 1 L raw sewage and 10 L treated effluent samples were performed using skimmed milk flocculation procedure and glass wool adsorption elution technique, respectively. For secondary viral recovery, both environmental samples were subjected to polyethylene glycol/sodium chloride precipitation. Manual extraction was used to extract the nucleic acids from the virus concentrate with mengovirus (MV) used as an extraction control. For the quantification of HAdV, standard curves prepared from known dilutions of HAdV and MV were used. Human adenovirus was detected in 140/150 (93%) of the environmental samples comprising of 69/75 (92%) being raw sewage and 71/75 (95%) being effluent samples. The HAdV concentrations detected in wastewater treatment plant 1 (WWTP 1) ranged from 1.38x105 gc/L to 4.50 x 109 gc/L for raw sewage and 5.08x103 gc/l to 4.30x108 gc/L for effluent. The HAdV concentrations detected in WWTP 2 ranged from 6.84x104 gc/L to 1.69x1012 gc/L for raw sewage and 5.27x103 gc/L to 1.16x108 gc/L for effluent. The HAdV hexon amplification success rate from the nucleic acids was 43/140 (31%). Eighteen HAdV genotypes were successfully characterised using Sanger sequencing. The HAdV-D was the most predominant species in both WWTPs, follow by HAdV-B and HAdV-F. The HAdV-A and HAdV-E species were the least identified. Next generation sequencing identified four times as many genotypes as Sanger sequencing (77 different genotypes). The HAdV-D (types 8, 9, 13, 17, 19, 20, 23, 24, 28, 29, 32, 33, 36, 42, 44, 47, 49, 51, 56, 60, 62, 64, 67 and 81) and HAdV-B (types 2, 3, 7, 11 and 66) were the most predominant species followed by HAdV-F (types 40 and 41), HAdV-A (types 12 and 76), HAdV-E ( type 4) and HAdV-C (type 1). Testing wastewater treatment plants is advantageous as it allows for the detection and identification of HAdV types circulating in the surrounding communities. Due to the large number of species identified using NGS, it is the superior typing method and should be used for future studies. These include strains causing symptomatic and asymptomatic infections. Human adenovirus was detected at comparable frequencies in raw sewage and treated effluent wastewater, with slightly higher detection in effluent samples. However, the viability of these viruses is unknown and should be investigated in further studies. The detection of viruses in wastewater treatment plants are a public health concern as the treated effluent is discharged into rivers, which may be used by communities for domestic and recreational purposes. / Dissertation (MSc (Medical Virology))--University of Pretoria, 2020. / NRF, PRF / Medical Virology / MSc (Medical Virology) / Restricted