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.

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. / Thesis (Ph.D.)-University of Natal, 1990.

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. / Thesis (M.Med.Sc.)-University of Natal, Durban, 1996.

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.

Antral mucosal biopsies were examined microbiologically and histologically for the presence of Campylobacter pylori in 224 patients with upper gastrointestinal symptoms. One hundred and eighty three (83%) patients were found to harbour Campylobacter pylori in their gastric mucosa. Campylobacter pylori was strongly associated with the presence of histological gastritis (93%) and was detected in only 10% of 30 patients whose gastric biopsies showed normal histology. Endoscopically diagnosed duodenal lesions were more strongly associated with the presence of Campylobacter pylori than were gastric lesions (p<0.001). A variety of laboratory methods were evaluated to determine the sensitivity and specificity to detect the presence of Campylobacter pylori. Histology was the most sensitive and specific method to detect the presence of Campylobacter pylori. Although culture was highly specific, it was less sensitive than histology in detecting Campylobacter pylori in gastric antral mucosal specimens. The "conventional" gastric urease assay, although specific, needs be performed under controlled conditions (37°C) for optimal results. The "one-minute" urease assay was more sensitive than the "conventional" gastric urease assays and was highly specific. ELISA to detect specific-IgG antibodies to Campylobacter pylori was a moderately sensitive non-invasive method to detect Campylobacter pylori infection, but was non-specific. / Thesis (M.Med.)-University of Natal, 1988.

Microbiological and molecular epidemiological studies were conducted on 36 multi-resistant Haemophilus influenzae strains, isolated from paediatric patients, over a 26 month period (April 1986 to May 1988). The majority of strains (80,5%) had been isolated from blood and cerebrospinal fluid. More than 80% of isolates tested belonged to biotype II and 90% were of serotype B. Minimal inhibitory concentrations against 6 antibiotics (ampicillin, chloramphenicol, tetracycline, rifampicin, streptomycin and cefotaxime) confirmed the presence of multi-resistant strains. Resistance to rifampicin was confirmed in 6 (16,7%) strains. All strains were susceptible to cefotaxime. Ten transconjugants analysed with respect to their plasmid content were shown to harbour an identical 41 MDa plasmid. Restriction endonuclease digests of these plasmids with Eco R1 and Sst1 revealed almost identical restriction patterns. Outer membrane protein profiles of 19 strains revealed the predominance of one particular subtype. By combining the microbiological and molecular epidemiological findings, it is concluded that one strain of H. influenzae type b is responsible for the nosocomial acquisition of infections amongst paediatric patients. The implifications of these findings are discussed. / Thesis (M.Med.)-University of Natal, Durban, 1988.

Tuberculosis (TB) is one of the most prevalent infectious diseases worldwide and causes high morbidity and mortality, despite the widespread availability of effective antibiotics against most strains of Mycobacterium tuberculosis, which is the causative agent of TB. One of the primary reasons that hinder TB control is that many cases of active disease go undetected or are discovered late. This is, in large part, due to the relative insensitivity and limited specificity, amongst other limitations, of the current TB diagnostics tests. Moreover, M. tuberculosis infection can be asymptomatic and latent, or cause active disease. Therefore, an ideal or effective TB diagnostic needs to distinguish between these two states. The aim of this study was to develop novel diagnostic reagents for M. tuberculosis using phage displayed peptides and nucleic acid aptamers with a view to discerning latent from active TB. Using a linear (X12) and constrained (CX7C) phage display libraries, five rounds of selection (biopanning) were performed. Ten phage displayed peptides that bind to the mycobacteria surface were selected. These phage clones were identified using both random clone picking and high throughput (HTP) sequencing. A phage clone displaying the CPLHARLPC peptide was identified by HTP sequencing as the most enriched, representing 82.49% of the selected CX7C phage population. Further characterization showed that it bound better to different mycobacteria species, including M. tuberculosis, than the unselected phage library. Moreover, using surface plasmon resonance (SPR) technology, the chemically synthesised CPLHARLPC peptide was shown to bind M. tuberculosis H37Rv whole cell lysate and not non-mycobacteria lysates. In addition, using the systematic evolution of ligands by exponential enrichment (SELEX) protocol and SPR technology, 2'-Fluoro-pyrimidine-RNA aptamers were selected against the mycobacteria ESX-3 secreted protein, ESX-G. At least five aptamers were identified after five rounds of selection. Two of these aptamers, GH43 and GH78, not only bound EsxG with high affinities, KD 8.04 ± 1.90 nM and KD 78.85 ±9.40 nM respectively, but also preferentially bound EsxG better than the EsxA homologue. Taken together, these findings suggest that a combination of phage display, SELEX and HTP sequencing can be a useful tool for the identification of specific detection reagents that can bind to mycobacteria and its associated targets. These reagents could be exploited to develop alternative molecular probes for TB diagnostics. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2013.

HIV-1 is the most prevalent and notorious sexually transmitted pathogen locally, and constantly challenges our foundation of knowledge regarding the classical STIs. The ultimate objective of the syndromic management strategy was to reduce the load of sexually transmitted infections, and hence HIV transmission. This strategy is multifaceted and not only includes the recognition of symptoms by the patient and an effective treatment regime that comprehensively covers the possible aetiological agents for a defined syndrome, but also appropriate health seeking behaviour of infected individuals, recognition of syndromes by the health care worker, partner management (notification and treatment), behavioural counselling and condom promotion. Understanding the complexity of sexual networking and transmission dynamics is part of such a strategy. So, although the rationale and design of syndromic case management appears simplistic, it is by no means easy to implement / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2002.

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. / Thesis (Ph.D)-University of KwaZulu-Natal, Durban, 2010.