Microbiology and Infection Control

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A study of some aspects of senescence in embryos of zea mays L.
(1968) Berjak, Patricia.; Villiers, T. A.
Abstract available in PDF file.
The RH Factor : a clinical and fundamental study of its significance in ISO- and Auto-Haemolytic anaemias.
(1973) Vos, Gerhardus Hubertus.; Villiers, T. A.; Bain, Peter G.
No abstract available.
Morphology, membrane characterization and detection of a bacterium associated with ratoon stunting disease of sugarcane.
(1984) Pillay, Dorsamy.; Roth, G.; Oellermann, Rolf Alfred.
Ratoon stunting disease (RSD) of sugarcane was first recognized in 1944 in Queensland, Australia (Steindl, 1961). The disease occurs worldwide and causes significant yield losses, especially during drought. RSD produces no external symptoms except a non-specific stunting (Steindl, 1961). RSD, which was first recorded 1n South Africa in 1953 (Anon., 1960), causes a greater overall loss in yield than any other sugarcane disease in South Africa. Yields of sugarcane are reduced by 20% to 40% and the harvest of affected fields declines progressively with successive ratoons (Anon., 1980b). A virus was originally thought to cause RSD, but in 1973, a coryneform bacterium was implicated as the causal agent (Gillaspie et al., 1973; Teakle et al., 1973). In 1980, our laboratory reported the successful isolation and culture of a coryneform bacterium associated with RSD of sugarcane and was indicated to be the causal agent (Nayiager et al., 1980). The lack of a rapid diagnostic technique applicable to mass screening of sugarcane has hindered progress in the control of the disease. There are two types of commonly used diagnostic tests. One test depends on the evaluation of internal stalk symptoms which may require from two to twenty six weeks to develop (Gillaspie et al., 1966; Matsuoka, 1971; Schexnayder, 1960; Singh, 1969). However, these symptoms are not always present in RSD affected plants and similar symptoms can sometimes result from other causes (Steindl, 1961). The other test involves establishing the presence of the coryneform bacterium associated with diseased plants. The bacterium is visible under high magnification by phase-contrast microscopy (Gillaspie et al., 1973) or by electron microscopy (Teakle et al., 1973). Although identification by the latter methods requires little time, the technology involved severely limits the number of samples that can be examined. Recently, serological techniques have been used (Brlansky et al., 1982; Damann et al., 1977; Davis et al., 1980; Gillaspie, 1978b; Gillaspie et al., 1979; Harris and Gillaspie, 1978) but their success has been limited. Besides problems with diagnosis of the disease, the precise morphology and taxonomy of the causal organism is unclarified. The objectives of this research programme were, firstly, to characterize the cultured intact bacterium and its constituent membranes both ultrastructurally and immunologically, and secondly, to evaluate various immunological methods for detection of the bacterium. This study should contribute to enhancing the taxonomic status of the bacterium and to the use of a rapid diagnostic technique applicable to mass screening of sugarcane. Such a technique should eventually contribute to effective control of RSD.
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.
A study of the prevalence of campylobacter pylori in patients with upper gastrointestinal symptoms, and an evaluation of various laboratory methods to detect its presence.
(1988) Miller, N. M.; Van den Ende, Jan.
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.
Microbiology and molecular epidemiology of multiresistant haemophilus influenza type B in Durban, South Africa.
(1988) Peer, Abdool Kader Cassim.; Van den Ende, Jan.; Smith, Arnold L.; Ward, Joel I.
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.
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.
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.
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.
Some aspects of liver disease in Black patients.
(1990) Maharaj, Breminand.
A study of the causes of liver enlargement amongst black patients at King Edward VIII Hospital, Durban, South Africa has revealed that congestive cardiac failure (36.7%), amoebic liver abscess (7.1%), hepatocellular carcinoma (5.8%) and cirrhosis (5.4%) are the most common causes in this population. Liver biopsy was needed to determine the cause in 28.7% of patients studied. The diagnostic yield of percutaneous liver biopsy was increased by obtaining 2 or 3 consecutive specimens for histological examination by redirecting the biopsy needle through a single entry site. This benefit was achieved without an increase in morbidity or mortality. Fatalities and complications associated with liver biopsy were more frequent at this hospital than in hospitals in Europe, The United Kingdom and North America. The complication rates after percutaneous or peritoneoscopic biopsy were 2.0% and 2.3% respectively. A total of 6 deaths was recorded. The morbidity and mortality rates were not increased when more than one specimen was taken during percutaneous biopsy. In the majority of patients in whom biopsy was carried out, after-care was either non-existent or inadequate. The "Tru-Cut" needle was used for all percutaneous liver biopsies at King Edward VIII Hospital. Two techniques, including the method recommended by the manufacturer, have been found to be incorrect; the needle must be used correctly if an adequate biopsy specimen is to be obtained for histological examination and if serious complications are to be avoided. Hepatic tuberculosis was diagnosed in 9% of patients with unexplained hepatomegaly who were subjected to liver biopsy. This disease did not yield any consistent clinical findings. In addition, liver function tests were of little diagnostic value and results of hepatic imaging techniques were often normal. Accordingly, a high index of suspicion is needed and liver biopsy is essential in patients with unexplained hepatomegaly or hepatospienomegaly, or pyrexia of unknown origin since biopsy provides the only means of diagnosing hepatic tuberculosis. The accuracy of both ultrasonography and scintigraphy in distinguishing between normal and diseased livers was low (68% and 74% respectively). These techniques performed better at detecting focal than diffuse liver disease; the sensitivity of ultrasonography and scintigraphy in focal and diffuse disease were 88% and 92%, and 27% and 54% respectively. The specificity of both procedures was high for both types of liver disease (range 91-96%). Overlap between the ultrasonographic features of amoebic liver abscess, hepatocellular carcinoma and metastatic carcinoma resulted in a correct final diagnosis being made in only 81% of patients with amoebic liver abscess, 29% with hepatocellular carcinoma and 43% of patients with metastatic carcinoma who had an ultrasound scan. Neither technique was capable of determining the cause of diffuse liver disease. Therefore, when diffuse parenchymal liver disease is suspected, liver biopsy is needed to determine the presence and nature of the disease. In addition, liver biopsy or aspiration is usually required to determine the cause of focal disease in selected patients in whom space-occupying lesions are detected on hepatic imaging studies.
Neutrophil cytoplasmic antibodies : their clinical associations and an improved method for their detection.
(1993) Duursma, June.; Pudifin, Dennis James.
The test for antineutrophil cytoplasmic antibodies (ANCA) was introduced into the author's laboratory in 1987. An improved indirect immunofluorescent method was developed, using a system which allows 16 instead of one serum sample to be screened on each microscope slide. The known disease associations of ANCA that have been explored include systemic vasculitis, renal limited vasculitis, chronic inflammatory bowel disease and HIV disease. In general the findings are similar to those which are emerging from other centres and confirm the value not only of the positivity but also the relevance of the intracellular disposition of the neutrophil cytoplasmic fluorescence in diagnosis. In this study 85% of patients with Wegener's granulomatosis were found to have C-ANCA. C, P and X-ANCA staining patterns were found in 57% of patients with ulcerative colitis. Forty one per cent of patients with symptomatic HIV have ANCA. Certain histological features such as neutrophil and vascular damage in invasive amoebiasis, and the established lytic effect of amoebae on neutrophils prompted the investigation of the possibility that ANCA may be generated in this disease. Seventy eight amoebiasis sera were screened and 98,70/0 gave a positive ANCA test with a pattern of fluorescence resembling that found in Wegener's granulomatosis. An ELISA test for specificity confirmed that, as in Wegener's granulomatosis, this amoebiasis-associated ANCA had proteinase 3 specificity. Of practical clinical importance is the fact that both HIV and amoebiasis are associated with a high level of ANCA positivity. These findings will need to be considered when ANCA tests are used in clinical decision making in an area where HIV disease and amoebiasis are endemic. A large number of normal volunteer blood donors have been tested and the false positivity rate of 0,5% confirms the specificity of the test.
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.
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.
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.
Microbiological aspects of enterococci isolated at King Edward VIII Hospital, Durban.
(1999) Pillay, Nithianandhi.; Sturm, Adriaan Willem.; Peer, Abdool Kader Cassim.; Bhamjee, Ahmed.
The increasing frequency of enterococci as a major cause of nosocomial infections and the transmission of these organisms amongst hospital patients demands a greater awareness of the Enterococcus. Therapy of enterococcal infections is complicated by the pathogens continually changing resistance patterns to many broad-spectrum antibiotics. In addition, the ability of enterococci to cause serious invasive infections including endocarditis and septicaemia with associated high mortality rates; prompted this study which was aimed at identifying the biological properties of enterococci isolated from blood cultures of patients admitted at King Edward VIII hospital, Durban. Enterococci were identified to species level by the API 20 Strep system which identified 68% and a conventional biochemical system of Facklam and Collins which identified 100% of the isolates.The emergence of beta-Iactamase producing enterococci in other countries encouraged the testing of all isolates for this enzyme. All were beta-Iactamase negative. The reported false susceptibility for aminoglycosides and cephalosporins with blood enriched media encouraged the testing of these antibiotics with and without the supplementation of 5% lysed blood. The results showed that an average false susceptibility of 55 % occurred for gentamicin and 35% for tobramycin and netilmicin. The cephalosporins affected, cefotaxime and cefuroxime showed a false susceptibility of 28% and 17% respectively. The choice of treatment for serious enterococcal infections is a syllergistic combination of a beta-Iactam antibiotic plus an aminoglycoside for enterococci with intrinsic low-level resistance. The development of high-level aminoglycoside resistance, MIC 22000,ug/ml results in loss of synergism. This study showed that 26.4 % of enterococcal isolates displayed high level aminoglycoside resistance i.e. to gentamicin and streptomycin. Time-kill study showed reduced killing rate for these organisms for the beta-Iactams and glycopeptides with low-level gentamicin resistance. The results confirmed that a cell-wall active agent combined with gentamicin can be successfully used for enterococcal therapy if the organism has intrinsic low-level resistance to this amino glycoside. Pulsed-field gel electrophoresis (PFGE) carried out on a selected number of Enterococcus faecalis and Enterococcus faecium with high-level aminoglycoside resistance showed a variability in the restriction endonucelase digestion patterns. This suggests independent development of high-level gentamicin resistance and not clonal expression. The ease and reliability with which enterococcal isolates may be typed using this technique to compare different strains represent a significant advance.
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.
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.
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.
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.
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.

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