In the 20 years since the pioneering publication of the genome of Mycobacterium tuberculosis, significant efforts have been made to complete functional annotation of the genome. However, these efforts have generally been performed on a single-gene basis, ensuring slow progress and leaving large portions of the genome unannotated. High-throughput approaches to understanding the functional genome, such as transposon-insertion sequencing, have been developed and applied to mycobacteria in a variety of conditions; however, they have several limitations, particularly in their ability to study genes essential for viability. The recent optimisation of inducible CRISPR-interference for mycobacteria offers the potential to expand the high-throughput functional genomic toolkit. This thesis utilises CRISPR-interference for the development and validation of two high-throughput functional genomic approaches in the model mycobacterium M. smegmatis. The first approach combines large-scale pooled oligonucleotide synthesis and nextgeneration sequencing, and is termed CRISPRi-Seq. A pooled library of 11 367 mutants, targeting 2 385 M. smegmatis genes with M. tuberculosis homologues, was constructed and used to infer gene essentialities which were compared with corresponding predictions from transposon-insertion sequencing data. This process validated the CRISPRi-Seq technique and identified practical considerations for its future use. The second approach utilises data derived from CRISPRi-Seq to create an arrayed library of 263 individual M. smegmatis inducible CRISPRi mutants targeting essential genes. This library is applied to a quantitative imaging pipeline to produce detailed data-driven profiles of the morphological impact of essential gene suppression. These morphological profiles are used to statistically predict genetic function, as well as antimicrobial mechanism-of-action. The two novel approaches developed in this work represent valuable technical advances and produce large datasets of functional genomic data which are available interactively online. Taken individually, or in combination, these methodologies can be utilised to increase fundamental understanding of mycobacteria, including the pathogenic M. tuberculosis

Infective endocarditis (IE) is a rare disease affecting heart tissues. The laboratory diagnosis of culture-negative endocarditis is complicated, and largely based on the combination of nucleic acid detection methods and serological investigation. There is a paucity of published data on microbes causing culture-negative endocarditis, but a recent report indicated that the bacterium Bartonella was the commonest cause of culture-negative endocarditis at a tertiary care facility in Cape Town, South Africa. This laboratory-based, non-clinical pilot study, evaluated the utility of a previously published real-time PCR assay for detecting Bartonella spp. on cats. This will be the first time this target will be evaluated in a real-time PCR assay to detect Bartonella spp. in human samples. For this study, we constructed a plasmid vector containing an insert of 83bp, derived from the Bartonella nuoG gene. In this non-clinical, laboratory evaluation, we used one laboratory sample to amplify the nuoG bacterial DNA fragment and cloned it into a plasmid vector. Using this plasmid in a technical validation, we demonstrated that the previously described assay could detect nuoG when using the LightCycler 480 Probes Master Mix. The results indicated that the assay reliably detected as little as 1000 copies of the target DNA, and infrequently also detected 10-100 copies of the target. The study showed no amplification using some commonly encountered organisms found in our clinical setting, thus indicating 100% specificity for Bartonella. We demonstrated that a plasmid construct containing an internal fragment from the nuoG gene successfully detected the target using a real-time PCR assay. Future testing should include further optimisation to improve reaction efficiency of the assay with spiked diagnostic samples, including peripheral blood, and DNA extracted from heart valve samples. The utility of the RT-PCR for diagnostic purposes should be evaluated by comparing assay turnaround time, sensitivity, and specificity of this assay versus the conventional PCR and Sanger sequencing currently in use to detect Bartonella spp. in heart valves. We concluded that the assay exhibited strong potential for use as a diagnostic PCR using the constructed plasmid, but that further optimization to improve PCR efficiency, and work to determine the clinical sensitivity and specificity are needed before the assay can be applied to blood samples.

Wheezing symptoms among children, present major health and economic problems globally. A recent study conducted in Canada observed a reduction in stool bacterial genera Faecalibacterium, Lachnospira, Veillonella and Rothia (FLVR) in three-month old infants with atopy-wheeze symptoms. It is not known whether this is true in different human populations worldwide. The overall aim of this dissertation was to investigate the contribution of any of the FLVR bacteria or their combination in the occurrence of infant wheezing within the Drakenstein Child Health Study (DCHS), South Africa. To address this aim, I began my thesis's project by conducting a systematic literature review which investigated the association of FLVR bacteria with the occurrence of different respiratory diseases in humans. My review provided evidence for the possible involvement of FLVR bacteria in human respiratory diseases, including asthma, pulmonary tuberculosis and pneumonia. Furthermore, this review highlighted the need for a well-designed and large study to investigate the contribution of the FLVR bacteria in respiratory diseases, in an African setting. Secondly, I optimized SYBR Green based real-time quantitative polymerase chain reaction (qPCR) as well as conventional PCR assays for the detection of FLVR bacteria. Using the optimized assays, I screened 533 stool samples collected from 140 wheezing and 140 nonwheezing infants. The optimized assays demonstrated good performance in the detection of FLVR bacteria from human stool samples. Using qPCR, Rothia, Veillonella, Faecalibacterium and Lachnospira were detected in 90% (479/533), 73% (388/533), 51% (274/533) and 14% (77/533) of the samples, respectively. Conventional PCR permitted the detection of Rothia, Veillonella, Faecalibacterium and Lachnospira in 55% (263/479), 74% (289/388), 53% (145/274) and 0% (0/77) of the qPCR positive samples, respectively. I also determined the factors associated with faecal colonization by FLVR bacteria in the first year of life. I showed that reduced colonisation by Faecalibacterium was associated with male gender (adjusted OR = 0.65, 95%CI: 0.42 - 0.98) and TC-Newman residence (adjusted OR = 0.52, 95%CI: 0.29 - 0.91). Breastfeeding was associated with less colonisation by both Lachnospira, (adjusted OR = 0.17, 95%CI: 0.05 - 0.49) and Veilonella (adjusted OR = 0.32, 95%CI: 0.10 - 0.91). Mother's tertiary education was significantly associated with high Rothia colonisation (adjusted OR = 11.73, 95%CI: 1.36 - 2.58). In the last section of my thesis, I assessed the association of FLVR bacteria with infant wheezing using logistic regression models. I found a significant association of Rothia with reduced risk of infant wheezing (adjusted odds ratio (aOR)=0.54, 95%CI: 0.28-0.93) and recurrent wheezing (aOR=0.29, 95%CI: 0.05-0.88). Using receiver operating characteristic curves (ROC), I showed that among all FLVR bacteria, Lachnospira (AUROC = 0.833, 95%CI: 0.64-1.00) and Rothia (AUC=0.707, 95%CI: 0.62-0.79) could serve as biomarkers for early prediction of infant wheezing. Overall, this is the first study on FLVR bacteria and infant wheezing to be conducted in Africa. Its findings encourage more research to be conducted in order to elucidate the potential protective role of Rothia against childhood wheeze and asthma, as well as the contribution of Lachnospira in asthma development.

The diagnosis of Mycobacterium tuberculosis by conventional techniques is associated with a number of problems which include lack of sensitivity (microscopy) and prolonged incubation periods (laboratory culture) . For these reasons various "rapid detection" methods have been developed but none of these meet all the requirements of sensitivity, specificity and rapidity. DNA probes, although rapid, only have a sensitivity similar to that of Ziehl-Neelsen stain. The polymerase chain reaction (PCR) has been shown to exhibit extreme sensitivity and specificity for a wide variety of infectious agents including virus, bacteria and protozoans. There have also been a few promising reports of PCR assays for M.tuberculosis and M.leprae. This thesis deals with the development of a PCR assay in which a repetitive 336 bp fragment is amplified from M.tuberculosis DNA. The assay is extremely sensitive (less than 10 organisms) and is entirely specific for M.tuberculosis. The assay was assessed in a comprehensive trial in which PCR was compared with conventional detection techniques in pleural fluids from 84 patients. The results of this trial demonstrate that PCR is more sensitive than laboratory culture and indicate that PCR can be used for the routine diagnosis for M.tuberculosis infections.

Although both are caused by Chlamydia trachomatis, Lymphogranuloma Venereum (LGV) presents differently from the infections caused by Oculogenital (OG) strains. The endothelium of blood and lymph vessels allows passage of cells to the site of infection. Endothelial cells also secrete chemokines and cell adhesion molecules which act as attractants and binding sites for various cellular immune components. Since LGV biovar affect the lymphoid tissue we studied the effect of C. trachomatis on endothelial cells. Human umbilical vein endothelial cells (HUVEC) were infected with C. trachomatis LGV serovars L1, L2, L3 and the OG strain E at multiplicity of infection (MOI) of 1 and incubated for 24 hours. Stimulation of Interleukin-8 (IL-8) and monocyte chemokine protein-1 (MCP-1) chemokines and the intercellular adhesion molecule -1 (ICAM-1) were quantified by enzyme linked immunosorbent assays (ELISA). Transendothelial migration of neutrophils and monocytes was carried out in transwells. The lactate dehydrogenase (LDH) release assay was used to measure cell necrosis. Apoptotic cell death was analysed using the BioVisionTM CaspGLOW Fluorescein Caspase Staining Kit and DeadEndTM Colorimetric Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) system with the C. trachomatis Culture Confirmation kit as a counter stain. All Chlamydia trachomatis serovars (L1, L2, L3 and E) successfully infected and replicated in HUVEC after 24 hours of infection. Only L3 stimulated significantly higher production of IL-8, MCP-1 and ICAM-1 by HUVEC as compared to the negative control and mock-infected cells. However, the remaining LGV serovars (L1 and L2) and the OG serovar E showed no significant difference in the stimulation of IL-8, MCP-1 and ICAM-1 when compared to the controls. Comparison of LGV and OG serovars showed no significant difference between these two biovars in inducing production of IL-8 and MCP-1, but L3 stimulated ICAM-1 at a significantly higher level than E. There was no significant difference in the number of migrated neutrophils between untreated HUVEC, mock infected HUVEC and HUVEC infected with Chlamydia serovars. L2 and L3 had significally higher amount of migrated monocytes than the controls with L3 being the highest. L3 was the only serovar that had a significant level of cell death by necrosis. Apototic cells were observed in both uninfected and infected HUVEC which is due to normal cell turn over. None of the infected cells showed TUNEL positive nuclei. It can be concluded that L3 is more virulent than the other serovars during the first 24 hours of infection. Infection with C. trachomatis serovars does not seem to cause any cell death by apoptosis 24 hours post infection. The only cell death that occurs is by necrosis and only on serovar L3 infected cells. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2011.

No description available.

Yellow fever virus (YFV) virus is an arthropod-borne virus that causes viral hemorrhagic fever in humans in the tropical parts of both Africa and South America. The virus belongs to the family Flaviviridae, of the genus Flavivirus comprising of approximately 70 viruses. It is transmitted to vertebrates by the bite of an infected female mosquito, primarily the Aedes species. It is a re-emerging pathogen with case-fatality rates that can exceed 50% in humans. YFV can cause an acute febrile illness in humans which can progress to severe disease with hepatic and renal failure. The diagnosis of infection and testing of the immune status of vaccinees require reagents that are prepared in biosafety level (BSL) three and four facilities. Therefore the development of a recombinant antigen that does not require BSL three facilities for preparation and is safe to use, would have an important role in a diagnostic laboratory for detecting antibodies in infected individuals and vaccinees. Despite the availability of a live-attenuated efficacious vaccine, it is not recommended for immunocompromised individuals, thus development of new generation vaccines would have important public health implications. Identification and mapping of antigenic regions and viral epitopes is important for development of subunit vaccines and improved diagnostics. Subunit vaccines focusing on antigens that induce a protective immune response provide a safe approach to the development of vaccines against diseases causing severe and frequently fatal haemorrhagic fevers. The aim of this study was to identify immunodominant viral proteins that induce detectable antibody responses that could be used for developing diagnostic assays and to identify linear B cell epitopes on selected viral proteins. The complete open reading frame of the genes encoding the domain III (EDIII) region of the envelope protein, capsid (C) and NS4a proteins of YFV were amplified, from the 17D strain of YFV, by RT-PCR using primers specifically designed from sequence data retrieved from GenBank. Oligonucleotide primers were modified with BamHI and HindIII restriction enzyme sites that facilitated downstream cloning. Each amplicon was cloned into the pGEM®-T Easy cloning vector using T/A cloning. Each gene was rescued from the recombinant plasmid using BamHI and HindIII restriction enzyme sites and ligated into bacterial expression system, pQE-80L vector. In a previous study, the YFV EDIII gene was cloned into pQE-80L and expressed in JM109 Escherichia coli cells however extremely low yields were obtained. In this study the expression levels were improved using different cell lines and optimizing incubation conditions. An insoluble 13 kDa protein was expressed from the construct and confirmed by Western blot analysis. The protein was expressed with a 6 x Histidine tag that was used to facilitate purification using a Ni2+ column under denaturing conditions. Attempts to express the YFV C and NS4a proteins were not successful and expression was abandoned. In an attempt to improve solubility the YFV EDIII gene was excised from the pGEM®-T Easy vector and subsequently cloned into pCold TF bacterial expression vector. A ~65 kDa soluble protein was expressed from the construct and purified under native conditions. The functional activity of the recombinant antigens in ELISA was compared with whole cell lysate antigen prepared from cell cultures infected with YFV. The biological activity of the recombinant YFV pQE-80L-EDIII antigen was confirmed in immunoassays using serum samples from humans vaccinated with YFV vaccine. Positive sera failed to react in ELISA using pCold TF expressed antigen and this antigen was excluded from further assays. A total of 20/24 serum samples from human vaccinees collected at varying stages after vaccination reacted in an ELISA with the recombinant YFV pQE-80L-EDIII protein and 24/24 reacted in ELISA with whole cell lysate antigen. The EDIII region of the envelope protein was shown to be able to differentiate between West Nile Virus infection and YFV infection in a limited number of convalescent horse sera. The recombinant EDIII protein was used to immunize mice. Serum samples collected from the mice reacted against whole cell lysate antigen in ELISA and was shown to have neutralising antibodies using an in vitro neutralisation assay. Hence the EDIII region of the envelope protein likely induces an important protective immune response. Finally, bioinformatics was used to predict possible epitope regions and using peptide libraries spanning predicted sites, one potential epitopic region was identified in the EDIII protein. Putative epitopic and antigenic regions along the length of the C, NS4a and EDIII proteins of each strain were predicted using the BCPREDS and ABCpred software. In conclusion, the EDIII protein, an immunodominant antigen of YFV, prepared in this study has some potential for differentiation of flavivirus antibodies although it lacks sensitivity for routine diagnosis. A potential epitope, TGHGTVVMQ, from amino acid 21 to 29 on the EDIII protein was identified using bioinformatics and was shown to have reactivity against immune sera. The significance of this epitope needs further investigation. Finally the EDIII region of the YFV protein shows potential as a target region for vaccine development as shown for other flaviviruses but which has not previously been published for YFV.

Thesis (M.Dip.)--Cape Technikon, 1988. / Typescript (photocopy) Bibliography: leaf 117-148. Also available online.

Thesis (Masters Diploma(Technology )--Cape Technikon, Cape Town,1988. / Nosocomical or hospital-aqired infection, can be defined as an infection not present when the patient enters a hospital. It usually manifests itself seventy two hours after admission and sometimes it is not apparent until after the patient has been discharged. When the incubation period is unknown, any infection developing after admission to a hospital, may be classified as a nosocomical infection.

The aim of this work was to purify type-specific glycoproteins from herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) for diagnostic use. The most likely candidate for a type-specific glycoprotein of HSV-1 is glycoprotein C (gC), although it has recently been shown to contain some type-common antigenic determinants. HSV-1 and HSV-2 were produced in BHK-21 cells and labelled with either (³H)-glucosamine ((³H)-gln) or a mixture of (¹⁴C)-amino acids ((¹⁴C)-aa). Analysis of the radiolabelled products by analytical sodium dodecyl sulphate polyacrylamide gel electrophoresis (SOS-PAGE) and autoradiography revealed that in the HSV-1 infected cells the radiolabelled components were incorporated into viral specific proteins only, whereas in the HSV-2 infected cells they were incorporated into host cell proteins as well as viral proteins. Preparative polyacrylamide gel electrophoresis (Prep-PAGE) was used as an initial step in separating HSV-1 infected cell proteins labelled with (³H)-gln. Two cycles of Prep-PAGE were sufficient to produce solutions containing either glycoprotein B ( gB) or glyco- protein C (gC), which were free of other HSV-1 glycoproteins. However, these solutions still contained a number of non-glycosylated proteins. Two different techniques were utilized to remove the non-glycosylated proteins from the glycoprotein solutions. Hydroxylapatite (HAUltrogel) chromatography in the presence of sodium dodecyl sulphate (SDS-HTP) did not separate the different HSV-1 glycoproteins and was not satisfactory for removing the non-glycosylated proteins. Gel-bound lectin affinity chromatography using wheat germ lectin and Helix pomatia lectin was not successful in purifying the glycoproteins because the glycoproteins which bound to the lectins could not be eluted under normal conditions. Difficulties encountered in eluting the HSV-1 glycoproteins from the lectins may have been due to the sodium dodecyl sulphate (SOS) in which the proteins were solubilized. For this reason, the gelbound lectin affinity chromatography was repeated using HSV-1 membrane proteins solubilized in a non-ionic detergent, Triton X-100. Using material prepared in this way, several HSV-1 glycoproteins were bound by wheat germ lectin and eluted under normal conditions to yield glycoproteins which were purified with respect to nonglycosylated proteins.