Microbiology and Infection Control

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Aspects of the epidemiology of malaria in Natal Province, Republic of South Africa.
(1990) Sharp, Brian Leslie.; Van den Ende, Jan.
This study investigated aspects of the epidemiology of malaria in the Natal province of the Republic of South Africa. In this study the Collins English dictionary definition of epidemiology is used where it is defined as the branch of medical science concerned with the occurrence, transmission and control of an epidemic disease. Malaria has been a notifiable disease in the Republic of South Africa since 1958. Retrospective malaria case data from the Natal province as a whole was analyzed and the data from the KwaZulu and Natal areas of the province compared. Malaria cases were reported from 35 of the 65 magisterial districts in Natal province during the study period. In the Natal areas 91.5% of the cases were reported from eight districts and in the KwaZulu areas 96.4% of the cases came from three districts or as imports from Mozambique. The overall attack rate for both the Natal and KwaZulu areas using the total population figures for each area were very similar for the period 1986-1988 at 0.71 and 0.70 per 1000 head of population for the respective areas. The disease showed a distinct seasonal pattern in the KwaZulu areas with 86.9% of the cases being classified as indigenous and only 13.1% as imported. In the Natal areas, however, the seasonal pattern was not as marked and only 12.1% of the cases were recorded as indigenous and in excess of 82% as imported. Three species of the Anopheles gambiae complex were found to occur sympatrically in Natal province, namely: An. arabiensis, An. quadriannulatus and An. merus. Of these species An. arabiensis was found to occur at five localities during or after the notification of indigenous malaria cases from these areas. Due to the sympatric distribution of these species particular emphasis was placed on species identification and in particular the biting behaviour and control of An. arabiensis was investigated. The study found both morphological and behavioural differences between populations of An. arabiensis from those areas of the province with an intra-domiciliary residual insecticide vector control programme and those from the unsprayed areas. In the unsprayed areas the majority of the indoor resting An. arabiensis had fed on man whereas in the sprayed areas the majority of the indoor resting An. arabiensis were bovine fed. In the sprayed areas, however, the majority of the An. arabiensis caught leaving huts had fed on man. The percentage survival of bloodfed An. arabiensis caught leaving huts in the DDT sprayed area was in excess of 72%. The data strongly suggest that optimal control of An. arabiensis will not be achieved using the current control strategy of the annual application of intra-domiciliary DDT.
The assessment of humoral immunity in the vaginal mucosa of pregnant and non-pregnant women.
(2003) Omar, Momeen.; Sturm, Adriaan Willem.
Mucosal surfaces are prominent in the gastrointestinal, urogenital, and respiratory tracts and provide portals of entry for pathogens. The mucosal immune system consists of molecules, cells, and organised lymphoid structures intended to provide immunity to pathogens that impinge upon mucosal surfaces. The aim of this study was to assess humoral immunity in the vaginal mucosa and compare this immune response to a systemic response. The use of commercially available tampons provided a self-administered, pain free method for the collection of vaginal secretions. To standardise specimens, a total protein determination was performed on vaginal secretions and on sera. All subjects were screened for sexually transmitted infections (STIs) using conventional and deoxyribonucleic acid (DNA) amplification tests. Immunoglobulin levels in vaginal secretions and in sera were quantitated using a quantitative sandwich enzyme- linked- immunosorbent assay (ELISA). The immunoglobulin levels quantitated were analysed on the basis of pregnancy status and the presence or absence of an STI. Immunoglobulin results for serum showed a significant increase in IgG and IgA in women with an STI regardless of pregnancy (p< 0.001). This study showed a decrease in vaginal IgG and IgA in women with an STI. Non-pregnant women with an STI had significantly lower levels of IgG and IgA in the cervico-vaginal secretions as compared to the controls (p=0.002 and p=0.0002 respectively). This was also observed in pregnant women (p= 0.03 and p< 0.001 respectively). IgM levels were mostly too low to be detectable but showed a tendency to increase in vaginal secretions of women with an STI. Pregnancy did not have an effect on immunoglobulin levels except for IgA. The effects observed were due to the presence of an STI. All the STI pathogens studied displayed a similar effect on immunoglobulin levels. Bacterial vaginosis, however, appears to exert an effect specifically on lowering IgG (p=0.008) in vaginal fluid and increasing IgG levels (p=0.008) in serum. Once a more complete understanding of the mechanisms associated with the host defence of the vaginal mucosa is obtained, specific immunotherapeutic strategies can be developed. A greater knowledge of host defence factors specific to the vagina will provide insights into understanding susceptibility to opportunistic infections and STIs.
An assessment of synthetic landfill leachate attenuation in soil and the spatial and temporal implications of the leachate on bacterial community diversity.
(2008) Govender, Kamenthren.; Hunter, Charles Haig.
The temporal fate of selected parameters, including redox potential; pH; phenol; nitrates; sulphates; copper and zinc, of a young synthetic acetogenic phase landfill leachate was assessed by perfusing a series of sequential soil (Hutton) microcosms (arrays) at two hydraulic loading rates (HLR). We chose HLRs that were representative of areas in South Africa with typically elevated rainfall (Pietermaritzburg – HLRh) and one with relatively low rainfall (Kimberley – HLRl). Preliminary phenol, copper, and zinc adsorption investigations on gamma radiation sterilized soil and unsterilized soil revealed superior adsorption rates for each compound in the unsterilized soil. This revealed the importance of the biological component of soil in phenol, copper, and zinc attenuation in soil. The results presented in this thesis suggest that the HLR of leachate into soil arrays contributes to significant differences in the fate of the landfill leachate parameters mentioned earlier. In addition, we assessed the temporal and spatial succession of bacterial community diversity in each of the soil arrays by polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). Prior to this, we compared two soil DNA isolation techniques, the modified method of Duarte et al. (1998) (Bead Beat) and the commercial Mo-Bio UltraClean™ Soil DNA isolation kit (Kit). The DNA isolated by the Kit method was significantly superior regarding purity and absence of DNA fragmentation. However, the Bead Beat method produced a significantly higher yield per reaction before further purification with Wizard™ Clean-Up columns produced DNA extracts of similar purity at the cost of a significant reduction in DNA yield. The Kit method was chosen for future DNA isolation and PCR-DGGE based on the quality of the PCR amplicons generated from the Kit isolated DNA. PCR-DGGE was further optimized by comparing the efficiency and sensitivity of a silver stain against ethidium bromide. Silver stain generated DGGE gels with greater number of bands (species richness – S) and stronger band signal intensities. Captured DGGE fingerprints generated data that were subjected to the Shannon-Weaver Index (H’) and the associated Shannon-Weaver Evenness Index (EH) to measure the change in spatial and temporal bacterial diversity. There was a significant shift in S and H’ for both HLRs but a significant change in EH was only observed for HLRh. Furthermore, a temporal comparison of S and H’ between both HLRs revealed significant differences throughout the investigation. Canonical Correspondence Analysis (CCA) revealed spatial distribution of bacterial community diversity with depth. Effects of phenol concentration, redox potential, and pH of the effluent leachate on bacterial community diversity was tentatively assessed by three-dimensional graphical representation on PlotIT 3.2 software. Bacterial community diversity showed a decrease with elevated pH and phenol concentration along with decreasing redox potentials for both HLRs. While this study reveals the spatial and temporal dynamics of bacterial community diversity in situ, it provides important evidence with respect to: (i) the effects of rainfall / leaching rates (HLR) on spatial and temporal bacterial community succession; (ii) the importance of the biological component in natural attenuation; (iii) the ability of soil, previously unexposed to landfill leachate, to initiate natural attenuation of phenol and other leachate constituents; (iv) the capacity of PCRDGGE to fingerprint successional changes in bacterial community diversity, (v) and the potential to clone and sequence selected members of bacterial associations for future reference in environmental remediation strategies.
Biodeterioration of aluminium hot roll mill emulsions.
(1998) Ramsden, Peter John.; Wallis, Frederick Michael.
An in-depth study of the biodeterioration of the Hulett Aluminium hot roll mill emulsion, Prosol, was conducted. Samples of the emulsion in use at the hot roll mill were taken from various areas of the emulsion reticulation system in order to identify regions of highest microbial contamination. Standard plate count techniques and diagnostic procedures were employed to quantify and identify the microorganisms in these samples. In some of the highly contaminated areas of the emulsion reticulation system, microorganisms exceeded lxlO'CFUml'1 emulsion. A range of bacteria was identified which included members of the genera: Bacillus; Pseudomonas; Escherichia; Enterobacter; Sporosarcina; Micrococcus; Aeromonas; Chromobacterium and Desulfovibrio. Various fungi, including several yeasts, were also isolated and some of the filamentous spore-forming types were identified zsAspergillus spp.; Penicillium spp. and a Cladosporium sp. A visual scale was developed to assess the growth rate of the isolated microorganisms on a range of specific media containing various emulsion components as carbon and energy source. Although the results obtained by using this scale were not conclusive, a few biodegradable components were nonetheless identified. It was found that mixed cultures of the above microorganisms had a greater biodeteriorative effect on the emulsion than did any of the pure cultures when applied separately. This suggested complex microbial interactions were involved in the breakdown of the emulsion. A laboratory-scale model system representative of the Hulett Aluminium hot roll mill was designed and constructed to carry out a series of tests on unprotected and biocide-treated emulsions. A range of biocide concentrations were tested from which the minimum biocide inhibitory concentration was calculated. It was shown that microorganisms exposed to sublethal doses of the biocide Busan (active component glutaraldehyde) over a prolonged period of time, exhibited greater levels of tolerance and resistance to the biocide than did those microorganisms not previously exposed. It was deduced that less frequent, shock doses of biocide are more effective in the control and eradication of emulsion degrading microorganisms than are frequent, low level doses of the same biocide. In addition to the biocide studies, three imported so-called 'biostable' emulsions were evaluated as possible replacements for the susceptible Prosol. Of these three imported emulsions, two viz. HRF3 and Houghton Biostable were shown to be more resistant than Prosol to biodeterioration. After assessing the current hot roll mill management practices, a number of recommendations were made, including: the improvement of plant hygiene; education of the mill workers; improvement of emulsion monitoring; improvement of down-time management and improvement of biocide dosing regimes. Recommendations are also made for minimizing potential microbial growth in the new hot roll mill currently under construction at the Hulett Aluminium processing plant at Pietermaritzburg, South Africa.
Cationic liposome mediated transfection with/without a targeting component.
(2005) Singh, Ashika.; Naidoo, Richard.; Singh, Moganavelli.
The transfer and expression of genes in cells is an important technique for basic research and gene therapy of human disease. A model for gene therapy has been investigated making use of a transfection complex consisting of three components, the DNA i.e. the gene to be transferred and expressed; a gene delivery vehicle viz. a cationic liposome and a cell specific targeting ligand, asialoorosomucoid (AOM). Cationic liposomes are positively charged liposomes that have been prepared from synthetic lipids and have been shown to complex or bind to DNA via electrostatic attraction. They have shown potential as an efficient non-viral gene delivery vehicle in human gene therapy. In this investigation, a novel cationic liposome consisting of 3B [N -(N',N'-dimethylaminopropane)carbamoyl] cholesterol (Chol-T), dioleoylphosphatidylethanolamine (DOPE) and biotinylcholesteryl formylhydrazide was prepared and assessed as a mediator of DNA delivery in a mammalian cell culture system viz. the HepG2 cell line. The cationic liposome was synthesised and characterised by electron microscopy. Foreign DNA may be specifically delivered to target cells by a carrier system which makes use of the recognition of the asialoglycoprotein AOM by cognate receptors on the HepG2 cell plasma membrane. The positively charged AOM was biotinylated and due to this biotinylation, binds streptavidin which contains specific binding sites for biotin. The cationic liposome itself contains biotin residues in its bi-Iayer which in turn binds streptavidin resulting in a ternary complex. Further, due to the DNA binding capability of the cationic liposome, a transfection complex is produced consisting of the three components. The experiments were based on the following concepts: (i) Hepatocytes possess a unique receptor that binds to and internalises galactose-terminal asialoglycoproteins by receptor mediated endocytosis. (ii) Due to electrostatic attraction, DNA binds to cationic liposomes forming soluble complexes. (iii) Through the biotin-streptavidin reaction, the biotinylated AOM is attached to the cationic liposome containing biotin forming complexes enabling targeted delivery of the DNA. (iv) DNA containing the pGL3 gene for the luciferase enzyme was used and following transfection experiments, the luciferase assay was performed to ensure successful transfection. The complexes were tested on the hepatocellular carcinoma cell line, HepG2, which possess the asialoglycoprotein receptor. Transfection studies were conducted using a transient expression system, the luciferase assay system. Some degree of success in the transfection of HepG2 cells was observed. Results obtained in this study suggest that transfection using our targeted transfection complex consisting of cationic liposomes and cell specific targeting ligands does in fact transfect cells by receptor mediation.
Characterization of 1, 2-DCA degrading Ancylobacter aquaticus strains isolated in South Africa.
(2011) Pillay, Thiloshini.; Pillay, Basil Joseph.; Olaniran, Ademola Olufolahan.
1,2-Dichloroethane (1,2-DCA), a highly toxic and recalcitrant compound, is produced anthropogenically in larger quantities than any other chlorinated compound. It is regarded as a mutagen and carcinogen, thus making it a priority target molecule for biological degradation. In addition, the intermediates of 1,2-DCA degradation are highly reactive and toxic, due to the electrophilic nature of the carbonyl groups in these compounds. Aerobic biodegradation of 1,2-DCA, resulting in complete mineralization, has previously been reported in Xanthobacter autotrophicus GJ10 and some Ancylobacter aquaticus strains. X. autotrophicus GJ10 has been found to possess chloroacetaldehyde (CAA) dehydrogenase and haloacid (HA) dehalogenase enzymes, both of which play a crucial role in 1,2-DCA degradation. Five strains of Ancylobacter aquaticus capable of utilizing 1,2-DCA as a sole carbon and energy source have recently been isolated in our laboratory. The degradation potential and specific dehalogenase activities of these bacterial isolates against 1,2-DCA and other halogenated compounds as a carbon source were investigated and compared to previously characterized organisms, viz., X. autotrophicus GJ10 and Ancylobacter aquaticus strains AD25 and AD27. Furthermore, this study proposed to detect the presence of the CAA dehydrogenase (aldB) and HA dehalogenase (dhlB) encoding genes in these isolates. Growth of all strains in the presence of 1,2-DCA as a carbon source was monitored over an 84 h period, in minimal medium supplemented with either vitamins or yeast extract. Dehalogenase activities were measured colorimetrically by monitoring halide release by crude cell extracts of the isolates. In order to detect the presence of dhlB and aldB genes, genomic DNA of the isolates was digested with individual restriction endonucleases, viz., EcoRI, PstI, HindIII and BamHI, and then subjected to Southern hybridization experiments. All isolates demonstrated significant growth rates in both vitamin and yeast extract supplemented media, with the former having a greater overall growth effect. Ancylobacter aquaticus DH5 demonstrated the highest growth rate of 0.147.h-1 in the presence of vitamins while Ancylobacter aquaticus DH12 displayed the highest growth rate of 0.118.h-1 with yeast extract. Optimum haloalkane dehalogenase activities of these bacterial isolates were confirmed at pH 8, similar to the activity in X. autotrophicus GJ10, while haloaciddehalogenase activity had a broader pH range. Hydrolytic dehalogenase activity of the bacterial isolates using a range of halogenated aliphatic compounds was also determined. Results demonstrated a wide substrate range with activity being observed on 1,3- dibromopropane, 1,2-dibromoethane and 1,3-dichoropropene, for all isolates. Southern Hybridization experiments confirmed the presence of both aldB and dhlB genes in X. autotrophicus GJ10. The dhlB probe produced a positive signal for an EcoRI fragment in Ancylobacter aquaticus DH12 while the aldB probe hybridized and produced a single positive signal on similar sized PstI fragments for all organisms except A. aquaticus AD25 which produced two positive signals. The results in this study demonstrate the potential application of the newly isolated strains of Ancylobacter aquaticus. in future bioremediation strategies. The detection of the genes involved in 1,2-DCA degradation further support the use of these isolates and/or their enzymes for the degradation of 1,2- DCA as well as other halogenated compounds. Future work need to determine sequence similarity of these genes detected in A. aquaticus strains to the genes in Xanthobacter autotrophicus GJ10 and other previously reported genes. It may also be important to investigate the activity of the enzymes under various environmental conditions and to determine enzyme structure and the catalytic sites, so as to gain knowledge of their degradation potential on site. Characterization of enzymes at both the molecular and protein levels may be necessary and beneficial for implementation in strategies involving bioremediation for the biological degradation of a wide range of halogenated aliphatic hydrocarbons.
Cloning of the endomannanase from Scopulariopsis candida LMK008 and evaluation of its effect on the digestibility on animal feed.
(2012) Gareeb, Ashant Pravin.; Govinden, Roshini.; Setati, Mathebata Evodia.
Present within the biodiverse hypersaline environment are a wide variety of halotolerant filamentous fungi. Many of these phytopathogens are capable of hydrolysing plant cell wall polysaccharides such as hemicellulose which are comprised of mannans and heteromannans which are polymers of the mannose sugars. Endoacting hydrolytic enzymes such as endo-β-1,4-mannanases are secreted into the extracellular environment and are involved in the catalysis of the random hydrolysis of β-1,4-mannosidic linkages within the backbone of mannan, galactomannan, glucomannan, and galactoglucomannan. Poultry are monogastric animals that are unable to efficiently digest high-fibre and mannan rich feeds such as soybean meals and this results in decreased or depressed animal performance. The use of feeds supplemented with β-mannanases has been shown to enhance the feeding value of mannan-based meals. In the current study, the degradation of β-mannan polysaccharides present in poultry feed by halotolerant Scopulariopsis candida LMK008 β-mannanase was investigated. SDS-PAGE, Native-PAGE in conjunction with zymogram analysis was used to assess the molecular weight of the endomannanases. At least three isozymes were detected: two of 56 kDa (pI 3.5 and 6.7) and one of 28 kDa. Anion exchange chromatography was used to purify the 28 kDa isozyme. Three mannan-based substrates, viz., locust bean gum, guar gum and soybean flour, were used to evaluate the hydrolysis capability of the crude as well as the purified β-mannanase via the release of reducing sugars and was detected using the DNS assay. The β-mannanase exhibited low activity with pure guar gum but high activity with locust bean gum galactomannan and soybean flour mannan. The hydrolysis activities of the crude and purified enzyme were then tested further on mannan-based soybean meals. In general it was found that more reducing sugars were released from the grower feed than the starter and layer feeds. Another common hydrolysis pattern observed in all feed types was that after prolonged incubation of 24 h there was a decrease in the amount of reducing sugars released which could be attributed to the presence of naturally-occurring microorganisms in the feed sample which metabolised the simple sugars resulting from the enzymatic hydrolysis of the mannan components in the feed samples. This was confirmed by standard plate count assays. The results obtained are encouraging and the purified β-mannanase could be applied as an industrial feed additive within the animal feed industry, however, further testing of the enzyme in situ is needed in order to prove its applicability. The cloning of the endomannanase has to date proven unsuccessful despite numerous techniques being employed and further research is also needed to accomplish this task.
A comparative epidemiological analysis of clinical strains of Staphylococcus aureus in Nigeria and South Africa.
(2005-09) Shittu, Adebayo Osagie.; Lin, Johnson.
Abstract available in PDF file.
Cryptosporidium and cryptosporidiosis.
(1990) Moodley, Dhayendre.; Jackson, Terry F. H. G.
Cryptosporidium parvum can cause debilitating disease in immunocompetent persons with cholera-like symptoms characterised by self-limiting, profuse diarrhoea; on the other hand asymptomatic infection with this organism frequently occurs. However, in immunocompromised patients, the disease is more severe and is lifethreatening. A pivotal aspect of the present survey was a comparative assessment of four commonly used staining techniques (viz. modified Ziehl-Neelsen, safranin-methylene blue, auramine phenol fluorescence and Sheather's sucrose flotation) for the detection and identification of Cryptosporidium oocysts. The Sheather's flotation method proved to be superior to the other three procedures which were not only less sensitive but also less specific. A modification of the Sheather's flotation technique was developed for use with diarrhoeal stools; this was found to be simple, reliable, costeffective and the least time consuming of the above methods; this was used exclusively in a subsequent survey of the association of Cryptosporidium infection with diarrhoea in hospitalised children. Although previous epidemiological surveys of cryptosporidiosis have been conducted in South Africa standardised methods have not been employed. This initial assessment of diagnostic techniques therefore provided a tool for accurately assessing the importance of Cryptosporidium as a causative organism of diarrhoea. In an extensive study performed on children younger than 10 years old, who were hospitalised with a primary diagnosis of diarrhoea at King Edward VIII Hospital, it was found that 9,0% (111/1229) were passing Cryptosporidium oocysts; this was the second most common enteric pathogen. In 72% (80/111) of patients with Cryptosporidium infections it was the only pathogen. The prevalence of cryptosporidiosis was highest during the months of February, March, April and May; direct correlation between the rainfall in the Durban area and the prevalence of cryptosporidiosis was demonstrated (r = 0,6125). Cryptosporidium infection was more prevalent in the 4-6 month age group (p = 0,001). The fact that Cryptosporidium infections may be symptomatic in some individuals and asymptomatic in others, suggests that strain differences in respect of pathogenic potential may occur. A prerequisite to the investigation of strain differences was to increase parasite numbers; both in vivo and in vitro culture techniques were employed. Culture in chicken embryos failed to increase the parasite population and only limited areas of the chorio-allantoic membranes showed a few developmental stages. Cell cultures proved to be more suitable for Cryptosporidium growth and parasite numbers increased proportionally with duration in culture. Attempts at infecting suckling Balb/c mice were unsuccessful; however experimental infection of immunosuppressed adult rats facilitated the examination of various developmental stages of the parasite. Isoenzyme electrophoresis is an excellent method for demonstrating polymorphism in many species. Of the five enzyme systems that were tested, glucose phosphate isomerase, malic enzyme and phosphoglucose dehydrogenase proved to be the most promising. The electrophoresis of lysates, prepared from oocysts, in an agarose gel system was found to give adequate and reproducible resolution of isoenzyme patterns. Isoenzyme polymorphism could be demonstrated in oocysts harvested from the stools of four children. Such polymorphism has not been described previously and indicates a more extensive study to investigate strain differences, and to correlate these with the clinical histories of infected subjects. This approach may be invaluable in elucidating the pathogenesis of Cryptosporidium infections in man.
Detection of carbapenem-resistant Enterobacteriaceae amongst neonates in a regional hospital in KwaZulu-Natal : screening for carbapenemase production and MIC correlation.
(2014) Govender, Kathleen.; Moodley, Prashini.
Carbapenemases are the primary cause for the increase in carbapenem resistance in Gram- negative Enterobacteriaceae. These enzymes are β-lactamases and have the ability to hydrolyse almost all β-lactam antibiotics thereby inactivating carbapenems that are used for the treatment of severe nosocomial infections. Multiple CPE outbreaks and epidemics have been reported in several hospitals in South Africa since the year 2011. This resulted in an increase in the morbidity and mortality rates and are slowly disseminating globally among more vulnerable individuals including neonates. Therefore, the aim of the study was to determine appropriate techniques for the rapid detection of carbapenem-resistant Enterobacteriaceae (CRE) (including CPE) isolated from neonates from King Edward VIII Hospital as well as to determine the molecular mechanisms conferring carbapenemase production in this subset of isolates. A total of 94 Klebsiella pneumoniae and 41 Enterobacter cloacae samples were isolated in this study. Among these species 10 % (9/ 94) and 39 % (16/ 41) of K. pneumoniae and E .cloacae respectively, were resistant to the carbapenems based on the Kirby-Bauer susceptibility tests, microbroth-dilution and E-tests. However, screening for carbapenemase production using chromogenic agar (Brilliance™ CRE agar and ChromID® CARBA agar), Modified Hodge test and amoxycillin-clavulanate double disc synergy test did not correlate with these resistance patterns and exhibited false positive results possibly due to the presence of extended spectrum beta-lactamase (ESBL) production by these organisms. Due to such discrepancies in the phenotypic results, further detection for the presence of carbapenemases was performed using multiplex real-time PCR assays. This revealed the presence of the blaOXA-48 gene in only 1 K. pneumoniae isolate. Further molecular characterisation will be required to determine if alternate mechanisms of resistance are present in the resistant isolates detected in this study.
Detection of drug metabolizing enzyme gene (DMEs) polymorphisms among the Zulu population of South Africa.
(2007) Makume, Mantha Thandiwe.; Naidoo, Richard.; Naidoo, Kogieleum.; Chelule, Paul Kiprono.
The ability to metabolise drugs and achieve positive therapeutic outcomes is dependent on both genetic and environmental factors. The focus of this study was to determine the distribution and frequency of clinically relevant DME alleles and to assess the impact of these DME alleles on therapeutic outcomes in a cohort of 50 HIV-TB co-infected Zulu participants. PCR-RFLP was used to generate a genotypic profile of CYPIA2, 2C9, 2C19, 2E1, 3A4, MDR-1 and NAT-2. The distributions of the allelic frequencies were as follows. The CYPIA2 (A) - 50.7%, CYP2C9*2 — 100% and *3 — 56.2%, CYP2C19*2 — 35.4%, CYP2E1 (C2) — 28.4%, CYP3A4*1B (G) — 58.2%, MDR-1 (C3435T) - 16% and NAT-2 slow acetylators — 6.5%. Seventy-three percent of participants had prolonged TB therapy. Within this group, 82.9% of patient displayed wild type and 17.2% variant allele for CYP2E1 gene (p= 0.04) profile. In addition, all the slow acetylators in this study had prolonged TB therapy. In the MDR-1 gene, 87.5% showed wild type allele and 12.5% displayed the variant allele. Unsuccessful TB outcomes were also noted in 22% of this study population. In this group the variant allele was found to be dominant in CYPIA2, CYP3A4 and NAT-2, the opposite was seen in CYP2E1 and MDR-1. It was also interesting to note a similar genetic profile in the group that showed successful TB therapy outcomes. All participants had positive ARV treatment outcomes despite DME genotypic variations. However, 26% of all study participants experienced liver enzyme abnormalities. These findings concur with other studies regarding the ethnic distribution of DME alleles and evidence of an association between ART and TB therapeutic outcomes and DME genotype variation was inconclusive.
The development and implementation of a mycobacterium tuberculosis rapid diagnostic assay, using reporter mycobacteriophages.
(2013) Makume, Mantha Thandiwe.; Sturm, Adriaan Willem.; Jacobs, William R.
Abstract available in PDF file.
Effects of management practices on soil organic matter content, soil microbial activity and diversity in the KwaZulu-Natal midlands.
(2002) Nsabimana, Donat.; Wallis, Frederick Michael.
The objective of this study was to investigate the effects of land use and management practice on the soil organic matter content and the size, activity and diversity of the microbial biomass. These effects were investigated using samples taken from the top (0-10 cm) layer of the soils from long-term agricultural managements including natural grassland, maize under conventional (maize CT), maize under zero tillage (maize ZT), annual ryegrass, Eucalyptus, Pinus, and permanent kikuyu pasture. The natural grassland was used as a control since records indicated that no agricultural activity had ever been exerted on the soil. The measurements used to investigate these effects included soil organic C, total N, soil pH, microbial biomass C, basal respiration rate, microbial quotient, metabolic quotient, dehydrogenase activity, fluorescein diacetate (FDA) hydrolysis, arginine ammonification rate, arylsulphatase activity and acid and alkaline phosphatase activities. The microbial functional diversity was measured using the Biolog Ecoplate and catabolic response profiles methods. Soil organic Cand total Nwere lowest under maize CT, followed by maize ZT and annual ryegrass and were higher under natural grassland, Eucalyptus and Pinus plantations while permanent kikuyu pasture had the highest values. The other analyses, namely microbial biomass C, basal respiration rate, FDA hydrolysis, arginine ammonification rate and arylsulphatase activity also followed the same pattern. Annual cultivation was responsible for a decrease in microbial biomass C, basal respiration rate and enzyme activity, principally because there was an appreciable decrease in soil organic matter content. Conversely, permanent pasture, Eucalyptus and Pinus plantations increased appreciably the amount of organic C and consequently, promoted the size and activity of the microbial biomass in the soils. The principle component scores showed that management practices affected the microbial functional diversity because different treatments were found in separate zones of the principle component spaces. The regression analysis showed that the variation in the PC1 and PC2 scores was correlated with the variation in soil organic C, exchangeable acidity, extractable P and exchangeable K and Mg. In addition, richness, evenness, Shannon, and Simpson diversity indices showed that any management practice affects the dynamics of soil microbial diversity.
Evaluation of a measles immunisation campaign in Natal/KwaZulu.
(1990) Abdool Karim, Salim Safurdeen.
Routinely collected data on vaccines supplied and administered, measles notifications and hospital admissions for measles were used to evaluate the 1990 measles immunisation campaign in Natal/KwaZulu. comparisons of the monthly averages during the 12 month period before the campaign, 4 months of the campaign and 12 months after the campaign indicated that the 1990 measles campaign in Natal/KwaZulu demonstrated that the campaign was limited, not by design, to blacks only. The campaign galvanised a high degree of participation from almost all health services in this region and resulted in a rapid and marked plunge in the incidence of measles as reflected by declines in both measles notifications and measles hospital admissions. There was no deleterious shortterm residual effect of the measles campaign on routine measles immunisation services. The spillover effects of the measles campaign on routine immunisation services against polio, tuberculosis and tetanus was generally beneficial. While the campaign was a success in generating involvement of health services in Natal/KwaZulu and reducing the burden of measles in this region, this disease has not been eliminated. Vigilance and continued routine vaccination efforts are required to prevent further epidemics of measles in Natal/KwaZulu.
Evaluation of laboratory methods for susceptibility testing of staphylococcus aureus.
(1988) Jansen van Rensburg, Hermanus Christoffel.
The susceptibility of 80 StaphyIococcus aureus isolated to oxacillin was investigated using microtitre, agar dilution and Stokes' disc diffusion methods. There was a bimodal distribution of the isolates according to the oxacillin minimum inhibitory concentration (MIC) values. For the sensitive isolates, the agar dilution method generally gave lower MIC values than the microtitre method, while for the resistant isolates the agar dilution method gave comparable to slightly lower MIC values than the microtitre method. The Stokes disc diffusion method yielding the best results when performed on Mueller-Hinton agar incubated at 30°C for 18 hours; however local strains grew poorly when incubated at 30 C for 18 hours. The next best medium which provided clear disc diffusion results plus good growth was Mueller-Hinton agar incubated at 35°C for 18 hours, on which 10 % of the sensitive isolates appeared intermediate in susceptibility, and none resistant, while all the resistant isolates (microtitre MIC >8mg/1) appeared resistant. Oxacillin resistance among strains of Staphylococcus aureus tested by Stokes' disc diffusion method correlated best with gentamicin resistance, and less often with tetracycline resistance. Therefore gentamicin- or tetracycline-resistance may indicate oxacillin resistance in Staphylococcus aureus.
Gene expression analysis of squamous cell carcinoma of the oesophagus using a novel real time PCR probe system
(2010) Malik, Neelam.
Squamous cell carcinoma of the oesophagus (OSCC) is a common malignancy that occurs with high frequency in certain parts of the world, including South Africa. The aetiology of OSCC has remained unclear although many studies suggest that it is caused by a combination of variable risk factors. Recent reports implicate a variety of genetic factors in the carcinogenesis of OSCC but their involvement is yet to be defined.
HIV epidemic types and customized prevention responses.
(2008) Abdool Karim, Quarraisha.; Hassanally, Leya.
Since the first reported case of AIDS 27 years ago, more than 70 million people have been infected with HIV worldwide, and 25 million have died of AIDS. The Joint United Nations Programme on HIV/AIDS (UNAIDS) estimates that 33 million people are currently living with HIV. The global prevalence of HIV has leveled off, but the total number of people living with HIV continues to grow.1 This article briefly discusses the roles that different HIV transmission modes play in the worldwide pandemic, and explains how the global pandemic is actually a complex mosaic of dynamic epidemics within and between countries.2 Further, it discusses matching appropriate prevention responses to two different types of epidemics: generalized and concentrated.
A hospital outbreak of multiresistant haemophilus influenzae type B.
(1996) Sattar, Kalawathie.; Hoosen, Anwar.
Following an outbreak of multi-resistant Haemophilus influenzae type b (Hib)infections in a tuberculosis hospital, this study was undertaken to determine carriage of Hib in 2 paediatric wards; to characterise all isolates of Hib, determine their antimicrobial susceptibility profile and the antibody response of the children to a conjugate vaccine. Prior to and one month after immunisation, oro- and nasopharyngeal swab specimens as well as venous blood were collected from each child. Isolates were tested for /3-lactamase and chloramphenicol acetyltransferase (CAT)production, their MIC's determined by the agar dilution method and characterisation of Hib isolates was performed by biotyping and analysis of outer membrane protein (OMP) profiles. An ELISA was also developed to determine serum antibody levels to polyribosyl-ribitol-phosphate (PRP), the capsular polysaccharide of Hib. The study population comprised a total of 135 children who had been hospitalised for treatment for tuberculosis. The patients were aged 4 months to 14 years with a median of 37,5 months. During the study period, none of the children developed invasive Hib disease. The overall carriage rate of Hib increased from 38% (51/135) before immunisation to 62% (84/135) after immunisation (P 0,15 /ig/ml. After immunisation, 34%(45) of patients increased their antibody levels to > 1,0 /xg/ml. There was no statistical difference between the mean antibody concentrations of patients who were colonised by Hib and those who were not (p = 0,58). The vaccine did not reduce carriage of Hib in this study population of children being treated for tuberculosis and the immune response to the vaccine was not optimal. Production of /3-lactamase and the prevalence of rifampicin resistance has implications for treatment and chemoprophylaxis in this population. OMP analysis showed a diversity of types. Multi-resistant strains causing invasive disease had the same OMP type as some multiresistant strains which colonised the children.
In vitro culture and isoenzyme analysis of giardia lamblia.
(1999) Kwitshana, Zilungile L.; Jackson, Terry F. H. G.; Sturm, Adriaan Willem.
Giardia lamblia, an enteric protozoan parasite, infects a large number of individuals worldwide. In South Africa prevalences ranging between 4 and 63% are documented, however, the impact of giardiasis is underreseached in this country. Giardia infections vary from asymptomatic carriage or a self-limiting acute symptomatic illness to chronic, debilitating malabsorption syndrome. The factors responsible for development of symptomatic versus asymptomatic infection are poorly understood. It is believed by some that host factors determine the clinical outcome of infection. On the other hand, the possibility of the existence of pathogenic and non-pathogenic strains (a situation akin to Entamoeba spp.) remains to be explored. One requirement for investigation of the potential contribution of strain differences to pathogenecity of infection is establishment of laboratory cultures of different strains isolated from symptomatic and asymptomatic patients. The present study was undertaken to develop and modify existing methods for: (i) establishment of laboratory cultures of Giardia trophozoites from excystation of faecal cysts, (ii) long-term maintenance and cryopreservation of the cultures and (iii) preliminary characterisation methodology. One thousand and twenty-three stool specimens were collected from day care centres, hospital wards and Hlabisa hospital laboratory. A further 6246 were retrieved from the Microbiology Laboratory at King Edward VIII Hospital and screened by direct wet preparation. Giardia was detected by light microscopy following formol-ether concentration (127 of 1023 samples) or direct examination of wet preparations (78 of 6246 samples). Cysts were purified from the positive specimens by sucrose gradient separation. Viability was assessed by a dye-exclusion method (eosin). Three in vitro excystation techniques were employed in an attempt to obtain trophozoites for initiation and establishment of viable cultures thereof. Culture conditions were optimised using two reference strains of Giardia, WB & H7 (obtained from the National Institutes of Health, USA). The percentage excystation ranged between 0-42% with all the in vitro methods of excystment. Excysted trophozoites remained viable in TYI-S-33 culture medium for periods ranging between 12-72 hours or up to 9 days, and gradually died, hence viable trophozoite cultures could not be established. Some culture initiates (overall 65%) were lost through overwhelming bacterial and!or fungal contaminants. An animal model was subsequently set up in which C57BL/6 and Praomys (Mastomys) coucha mice were used for in vivo excystation experiments. 1-3 day old suckling mice were intragastrically injected with 10,5 -cysts/ ml in 0,1 ml distilled water. Trophozoites were retrieved from the stomachs of infected mice 7-10 days after inoculation and cultivated in TYI-S-33 medium. Six local isolates were axenised using the in vivo excystation method. They have been maintained for more than 15 months in culture after stabilates and Iysates of confluent growths had been cryopreserved in Liquid Nitrogen. Successful (100%) retrieval of the cryopreserved cultures has been achieved. Seven isoenzyme electrophoresis systems have been set up and optimised. Reproducible results were obtained in six of the enzymes. Some differences in banding patterns of the enzymes were demonstrated.
The interaction of lymphogranuloma venereum and oculogenital chlamydia trachomatis with human keratinocytes and cervical epithelium.
(2010) Joubert, Bronwyn C.; Sturm, Adriaan Willem.
Background. Keratinocytes are the first target of infection for lymphogranuloma venereum (LGV) Chlamydia trachomatis, yet they have been omitted from pathogenesis studies. We infect keratinocytes and cervical cells with C. trachomatis and hypothesize different growth and cytotoxicity profiles among the strains. Methods. HaCaT human keratinocytes and ME-180 cervical cells were infected with C. trachomatis (multiplicity of infection (MOI) 0.025) serovars L1, L2, L3, 3 LGV clinical isolates or serovar E and incubated at 37 or 33°C for 5 days. Cytotoxicity was quantified daily using the CytoTox96® Non-Radioactive Cytotoxicity Assay, cells stained with the MicroTrak C. trachomatis Culture Confirmation kit and growth quantified by area of 100X photographs covered by Chlamydia. HaCaT and ME-180 cervical cells were infected with C. trachomatis (MOI 0.25) serovar L2 or E, incubated at 37 or 33°C for 48 hours and viewed with a transmission electron microscope (TEM). Mitochondrial activity was quantified using the MTT assay. The DeadEndTM Colorimetric TUNEL System with C. trachomatis Culture Confirmation kit as a counter-stain was used to assess cell death in infected versus uninfected cells. The BioVisionTM CaspGLOW Fluorescein Caspase Staining Kit and Transwell® Permeable Supports was used to differentiate between apoptosis mediated by cell-to-cell contact or a secreted molecule. Results. Growth in ME-180 versus HaCaT cells at 37°C was similar, but slower at 33 versus 37°C in HaCaT cells (p < 0.05). By day 5 L2 had grown faster than other strains in HaCaT cells at 37°C (p < 0.05), faster than clinical isolates in ME180 cells (p < 0.01), and faster than serovar E, and 2 clinical isolates at 33°C (p < 0.01). After 5 days L2 induced cytotoxicty was 11% in ME180 cells, which was higher than the clinical isolates (p < 0.01). In HaCaT cells at 33°C L2 EB were identified in a non-membrane state in the cytoplasm but not in the inclusion at 48 hours post infection. Serovar E but not L2 caused mitochondrial swelling at 1 h post infection in HaCaT cells at 37°C. This corresponded with a 16% reduction in mitochondrial activity (p < 0.001). TUNEL assay analyses demonstrated numerous dead cells adjacent to chlamydial inclusions for strains L2 and L3 but not L1 and E. An elevated number of caspase positive cells was detected in uninfected cell monolayers exposed to both L2 and E at 37°C but not 33°C. Conclusions. 1. C. trachomatis infects human keratinocytes in vitro. 2. Fresh clinical isolates behaved differently to the L2 reference strain. This demonstrates the need for fresh clinical isolates in pathogenesis studies of LGV. 3. In HaCaT cells at 33°C serovar L2 EB leave the intact inclusion and migrate through the cytoplasm in a non-membrane bound state 4. C. trachomatis induces apoptosis in uninfected cells exposed to infected cells via a secreted molecule at 37°C. This is more marked with serovar L2 exposure than serovar E exposure.

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