With an estimated third of all tuberculosis (TB) cases being missed, the need to develop rapid, simple and accurate diagnostic tests is critical. The last five years has seen an unprecedented activity in the development of a range of new tests. However, a major concern is that not all marketed TB tests have been assessed rigorously, particularly in terms of diagnostic accuracy, robustness under operational conditions in the field, and practical usefulness. This dissertation comprises a compilation of diagnostic clinical studies of novel point-of-care tests, namely a chemiresistive "TB breath-analyser"; a lipoarabinomannan (LAM) urine dipstick, and an adaptation of the Xpert®MTB/RIF assay for use on blood. Lastly, there is a modification of the sputum collection device (SCD) to enable specimen processing without the requirement of a biosafety cabinet. The chemiresistive sensor, which detects volatile organic compounds released by Mycobacterium tuberculosis in a patient's breath, demonstrated a high sensitivity (100%) and specificity (92%) for distinguishing patients with active TB from healthy controls. However, sensitivity (74%) and specificity (63%) were lower when the culture-negative participant group was compared to the culture-positive participants. The test shows potential as a useful screening test for TB with further refinement of the sensor technology. The LAM dipstick was shown to be useful in hospitalised HIV-infected patients with CD4 T-cell counts <200 cells/μL reinforcing the data from other studies. Although the blood Xpert®MTB/RIF assay showed some utility in diagnosis of TB in hospitalised patients with very advanced HIV, given the poor sensitivity and specificity, and the requirement for specialised equipment as well as a large volume of blood for testing, it is unlikely that Xpert®MTB/RIF testing on blood will contribute much over other existing diagnostics in resource-limited settings. Finally, the redesigned SCD offers a solution to biosafety concerns with minimal impact on patient acceptability and clinical care.

Tuberculosis (TB) is one of the top ten leading causes of death worldwide with millions of new TB cases reported every year. Understanding the genetic diversity of Mycobacterium tuberculosis (M. tuberculosis) is very crucial for rapid diagnosis and to reduce transmission of TB. Various diagnostic techniques, anti-tuberculosis reagents and vaccination are available, however, the disease is far from being eradicated (Brudey et al., 2006). Mycobacterium tuberculosis is classified into seven major lineages that are key to the most research areas. Recently, multidrug M. tuberculosis have been reported as the most dangerous strains that cause a life-threatening TB. However, the M. tuberculosis with modified virulence and transmissibility, particularly those that are caused by mutations leading to genetic variation and increased pathogenicity are highly reported (Zaychikova et al., 2015). Genetic markers such as variable number tandem repeats, insertion sequence element and direct repeats have been used to identify lineages. However, the techniques (such as spoligotyping, IS6110-RLFP and MIRU- VNTR) that use these genetic markers have a lot of drawbacks and some have low discriminatory power (Mikheecheva et al., 2017). Recently, single nucleotide polymorphisms (SNPs) are regarded as the most promising genetic markers for genotyping M. tuberculosis because they have low-level homoplasy and high discriminatory power (Zaychikova et al., 2015). The present study proposed that genotyping M. tuberculosis using polymorphisms in virulence genes may be an alternative approach to determine lineages and may help to detect the M. tuberculosis strains that are epidemiologically dangerous and have adapted to specific geographic regions. This study aimed to identify and evaluate a set of virulence gene SNPs as markers of M. tuberculosis strains circulating in the Tshwane region. A total of 150 susceptible and resistant M. tuberculosis cultures stored in Mycobacteria growth indicator tubes (MGIT) tubes were collected from May to October 2018 at the National Health Laboratory Service, Tshwane Academic Division (NHLS/TAD) to conduct this study. The DNA was extracted using hexadecyltrimethylammonium bromide (CTAB) method and spoligotyping was done to screen for M. tuberculosis lineages. The Beijing and LAM genotypes detected by spoligotyping were sequenced using the Illumina Miseq platform. The bioinformatic analysis of virulence genes in 56 genomes of M. tuberculosis belonging to Beijing and LAM genotypes was performed to detect lineage-specific SNPs markers. Of the 150 M. tuberculosis collected, 57.3% were susceptible M. tuberculosis strains while 42.7% were drug-resistant TB. Spoligotyping of 150 isolates resulted to 86.7% previously shared type (ST) and 13.3% orphans yielding a clustering rate of 63.3%. The Beijing family was found to be the most predominant lineage by 26.7%, followed by T family (16%), LAM (13.3%), East Africa Indian (EAI) (8.7%), S (6%), Manu (4.7%), H (4.7%), CAS (4.0%) and X3 (2.7%). The number of susceptible M. tuberculosis isolates per lineages was higher than drug-resistant TB with isolates detected as Beijing contributing 17.3% of all susceptible isolates, followed by isolates classified as orphans (10%), T family (9.3%), LAM family (8%) and CAS (2.67%). The association between anti-tuberculosis drug-resistant TB and lineages was found in EAI lineage (6.7%), Manu (4%) and S family (3.3%). The family with a high number of isolates which were drug-resistant TB was the EAI1-SOM sub-lineage belonging to the EAI family. This study successfully identified 29 Beijing and 6 LAM signature SNPs that can be used to classify clinical M. tuberculosis isolates. Within these signature SNPs, fadD28 (1521 C>T), eccCb1 (1479 G>A), pks5 (6210 G>A), and ponA2 (372 G>T) were identified in the Beijing strains and fadD28 (1392 C>G) within the LAM strains that were not reported in previous studies. Furthermore, this study detected the lineage-specific SNPs: mce3B (145 T>G), eccCb1 (1556 G>T), vapC12 (95 A>G) in Beijing BO/W148 and cyp125 (1076 T>C), mce3B (44 T>C), vapC25 (221 A>C), vapB34 (140 C>A) F15/LAM4/KZN sub-lineages which have been reported to be virulent and associated with drug resistance. This study showed a high genetic diversity of M. tuberculosis strains circulating within the Tshwane region. The Beijing lineage identified in this study was found to be more predominant than the rest of the identified genotypes. This study proposed the alternative method for genotyping M. tuberculosis strains using SNPs in virulence genes of M. tuberculosis. Observations from this study also highlight the advantage of using WGS technique over other genotyping methods such as IS6110-RFLP that has more drawbacks, as most genotypic methods discriminate M. tuberculosis strains using specific genes or regions in the genome of M. tuberculosis while WGS uses the complete genome of M. tuberculosis to determine different M. tuberculosis lineage. / Dissertation (MSc)--University of Pretoria, 2020. / The thesis/Dissertation is under embargo until September 2023. / National Research Foundation (NRF) / The thesis is under embargo until September 2022. / Medical Microbiology / MSc / Unrestricted

Background Mycobacterium tuberculosis isolates such as the Beijing and F15/LAM4/KZN families dominate in patients. The emergence of extensively drug resistant (XDR) M. tuberculosis isolates raises concern. The need to better understand the pathogenesis of M. tuberculosis isolates resulted in this work. Methods M. tuberculosis clinical isolates that belonged to the Beijing and F15/LAM4/KZN families, isolates with unique DNA fingerprints and laboratory strains were used. Isolates were grown in the presence of oxygen and then exposed to A549 alveolar and BBM bronchial epithelial cells. The number of bacilli that adhered to the epithelial cells were viewed and counted using light microscopy. Isolates grown in the presence of oxygen and under oxygen deprivation were used for subsequent assays. Invasion of A549 and BBM cells by isolates grown under these different circumstances was investigated. Based on the results, the remaining assays were performed with A549 cells only. Cytotoxicity was quantified using the Cyto Tox96 Non-Radioactive Cytotoxicity Assay kit. Morphological changes in A549 cells after exposure to the isolates were observed using the scanning electron microscopy (SEM). Real-time quantitative PCR was performed to assess the relative expression levels of four genes potentially associated with virulence (hbhA; mdp1; fdxA; hspX). Results were normalized against 16S rRNA and ftsZ gene transcription and reported as fold difference as compared to H37Rv. Results All isolates adhered to and invaded A549 cells in significantly higher numbers than BBM cells (P<0.0029). Isolates grown under oxygen deprivation displayed higher levels of virulence than their aerobic phenotype. Grouped together, the isolates belonging to the Beijing and F15/LAM4/KZN families of strains showed greater adhesion capacity (28%) than isolates with unique DNA fingerprints (5%) (P<0.05%). Three F15/LAM4/KZN isolates (two XDR-variants), were at least twice as invasive (>33%) as the most invasive Beijing isolate (15%) (P<0.05). The highest cytotoxicity level (35.7%) was produced by an XDR-F15/LAM4/KZN strain. SEM revealed bleb-like structures on bacterial cells grown under oxygen deprivation. Beijing and XDR-F15/LAM4/KZN isolates had the highest number of projections (16+5 per bacillus. The expression levels of all four genes were highest in Beijing and F15/LAM4/KZN isolates grown under oxygen deprivation and exposed to A549 cells. Conclusions Beijing and F15/LAM4/KZN strains are more virulent and their successful spread might be related to their interaction with alveolar epithelium. M. tuberculosis pathogenesis studies should include isolates grown under oxygen deprivation. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2013.

Treponema pallidum is the causative agent of the sexually transmitted disease, syphilis. The organism can not be cultured in vitro, which has inhibited the understanding of the pathogenesis of syphilis. There has been no evidence of a treponemal toxin but adherence of large numbers of treponemes is able to destroy cell monolayers of different cell types (Fitzgerald et al, 1982). Non-pathogenic treponemes failed to adhere to cultured cells and this suggests that adherence is associated with virulence of T. pallidum (Fitzgerald et al, 1977). In this study we explored the interaction of T. pallidum with HaCaT cells which are immortalized human keratinocytes with characteristics equivalent to their natural counterpart. The adhesion assay confirmed binding of the organism to HaCaT cell monolayers. Migration assays and electron microscopy revealed that T. pallidum migrates through a confluent keratinocyte layer and western blotting experiments that differentiate between soluble and insoluble occludin confirmed that T. pallidum does not loosen the tight junctions. It is concluded that T. pallidum passes through the keratinocyte layer by trans-cellular rather than inter-cellular migration. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2010.

Trichomoniasis has been linked to pelvic inflammatory disease, cervical cancer, increased HIV transmission, infertility as well as co-infections with other STIs. In 2002, an association was found with Trichomonas vaginalis and PID in HIV positive women. Therefore, the question arose whether T. vaginalis is able to invade the upper genital tract of HIV infected women. A prerequisite for invasion of the upper genital tract is the capability of the organism to adhere to the cells of the organs involved. This study therefore investigated the interaction between T. vaginalis and vaginal, cervical and endometrial cells. In comparing adhesion and cytotoxicity of T. vaginalis to cells of the upper and lower genital tract at different pH, immortalized vaginal (VK2), cervical (ME 180) and endometrial (KLE) cells were exposed to a standardized inoculum of trichomonads at pH 4.5 to 7.0. Adhesion was measured microscopically after acridine orange staining and cytotoxicity was established by measuring LDH release using a commercial kit. Adhesion of the ME-180 and VK2 cell lines was found to be pH dependent. However, the KLE cell line was not. As the pH increased, adherence to the vaginal and cervical cells decreased. Adhesion to endometrial cells was minimal at neutral pH but marked adhesion was found at lower pH. For the vaginal cell line, cytotoxicity was minimal at pH 4.5 but substantial (30 to 60%) at higher pH. In contrast, cytotoxicity on cervical and endometrial cells was highest at lower pH. The pronounced toxicity of vaginal epithelial cells at pH 5 and pH 5.5 is in keeping with the pH range found in patients with vaginitis. The observations on the cervical epithelium suggest toxic effect on the ecto-cervical epithelium immediate after acquisition of the infection. Adhesion of trichomonads to the endometrial cell line suggests that T. vaginalis is capable of colonization of the upper genital tract. At pH values applicable to the in vivo situation, toxicity was very low. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2010.

<p><em>Chlamydia pneumoniae </em>is a Gram-negative, obligate intracellular bacterium which shares its unique biphasic developmental cycle, genus-specific lipopolysaccharide, and complement fixation antigen with the other <em>Chlamydia</em> species. Intracellular bacteria, like <em>Chlamydia</em>, require strategies to invade host cells, evade host detection, commandeer host processes, and absorb nutrients in order to support their developmental cycle and survive. The type III secretion (T3S) system meets these needs by transporting bacterial effector proteins across the bacterial membrane and through the host cell membrane. The T3S system in <em>C. pneumoniae </em>is composed of approximately twenty different proteins, whose encoding genes are dispersed throughout ten operons in the <em>Chlamydia</em> genome. CdsD (<em>Cpn0712</em>), a basal body protein component of the T3S apparatus, is suggested to localize to the inner membrane and anchor other T3S structural components of the inner membrane ring. However, the cytoplasmic N-terminal domain contains two putative forkhead-associated (FHA) domains which may play an additional functional role in cellular signalling. This large hypothetical inner-membrane protein is poorly characterized in <em>C. pneumoniae </em>and the role of the predicted phospho-threonine binding, N-terminal FHA domains has yet to be elucidated. Herein, we provide evidence that CdsD has a high affinity for five cytoplasmic (CdsQ, CdsL, CdsN, PknD and SycH) and one periplasmic (CdsF) T3S-associated proteins. We also provide the first evidence that the phosphorylation of CdsD may permit the phosphorylation-dependent oligomerization or interaction with other phosphorylated components of the T3S apparatus. Future research will clarify the role of phosphate signalling in the T3S virulence mechanism. Ultimately, this may lead to a greater understanding of signalling mechanisms that regulate the secretion of bacterial effectors into host eukaryotic cells.</p> / Master of Science (MSc)

No description available.

University of Macau / Institute of Chinese Medical Sciences

Extended spectrum (3-lactamases (ESBLs) were characterized in Salmonella spp. isolates from a pediatric ward of a hospital in Durban. Forty one Salmonella spp. were subjected to serotyping, antibiotic susceptibility testing, E-Tests for ESBL detection, iso-electric focusing, polymerase chain reaction for detection of genes and sequencing. Isolates were screened for the presence of WaTEM, WaSHV, WaCTX-M, WaOXA , WaCMY, WaDHA and WaACC genes. The most common serotype was Salmonella Typhimurium. Isolates were multi-drug resistant with 100% susceptibility only to meropenem and ciprofloxacin. Tazobactam was the most effective inhibitor. Forty-one percent of the isolates were resistant to ceftriaxone, thus limiting therapeutic options for Salmonella infections.TEM-1 was the most predominant (3-lactamase found in 51% of isolates while SHV-12 found in 39 % was the most common ESBL. TEM-63 was evident in 29 %, TEM-116 in 10 % and TEM-131 was found in one isolate. The high ceftazidime MICs of isolates expressing only TEM-63 were indicative of R164S substitution which widens the binding cavity to accommodate the bulky side chains of oxyiminoaminothiazolyl cephalosporins. The identification of TEM-131 which differs from TEM-63 by 1 amino acid reiterates the evolutionary potential of the TEM-type plactamase. Other ESBLs identified included SHV-2, CTX-M-3, CTX-M-15 and CTX-M-37. CMY-2 and the OXA-1 p-lactamase were also detected. This is the first report of TEM-116, CTX-M-3, -15 and -37 in Salmonella spp. in South Africa. All isolates with nalidixic acid MICs > 48 ug/ml had the mutation D87N, or D87G in the QRDR of the gyrA gene. This study showed that Salmonella spp. may be multi-drug resistant with the propensity to harbour p-lactamases in unique combinations. The diversity of ESBLs and the co-expression of quinolone resistance suggests that their incidence in salmonellae needs to be monitored. / Thesis (Ph.D.)-University of KwaZulu-Natal, 2008.

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. / Thesis (M.Med.Sc.)-University of Natal, Durban, 1990.