Identification and drug sensitivities of Acanthamoeba species causing keratitis : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand

Acanthamoebic keratitis is a distinct, vision-threatening ophthalmological condition, the incidence of which is increasing with increased usage of contact lenses. Diagnosis can be difficult and chemotherapeutic treatment is prolonged and often ineffective. It is therefore desirable to discover a quick and accurate means of diagnosing acanthamoebic keratitis, and to gain knowledge regarding which chemotherapeutic agents are most effective against acanthamoebic keratitis. The first goal of this thesis was to examine ten DNA extraction procedures and determine their effectiveness in extracting DNA from Acanthamoeba cells. Of these ten methods, four (2, 7, 8, 10) could be performed in less than one day and showed consistent results in PCR reactions. The second goal of this thesis involved the application of arbitrarily primed polymerase chain reaction (AP-PCR) in an attempt to type and group strains of Acanthamoeba species. Examination of 16 isolates with primers AP0l, AP02, AM1, AM2, P1 and P2, showed each of the banding patterns, resulting from AP-PCR analysis, were unique to the isolate tested. Further, there were few bands which occurred in more than one isolate, with insufficient similarities to form groupings of isolates. Two chemotherapeutic agents were selected for a preliminary study into drug sensitivities in Acanthamoeba species and strains. The first of these was Baquacil (20% polyhexamethylene biguanide (PHMB)), and the second was Brolene (0.1% propamidine isethionate). Within 48 hours 97% of all isolates tested reached zero viability at a concentration of 0.05% PHMB, and 100% of isolates tested reached zero viability at 0.1% PHMB. The results of this study would suggest that the concentration of PHMB be at least 0.05% when used to treat acanthamoebic keratitis. Within 48 hours of exposure to 0.1% propamidine isethionate (Brolene), only 30% of all isolates tested reached zero viability. However, 60% of isolates tested showed at least 80% reduction in viability within 48 hours of exposure to 0.1% propamidine isethionate. The ultimate goal of this thesis was to form groups of isolates using PCR and drug sensitivities and to discover any correlation between these groups. The results of AP- PCR analysis however suggests a high genetic heterogeneity within the Acanthamoeba genus, thus preventing any correlation with drug sensitivity tests

Ocena kratkotrajnega raztapljanja in izločanja karbonata z uporabo vrstičnega elektronskega mikroskopa: metodološki postopki in preliminarni rezultati iz Postojnske jame

Carbonate dissolution and precipitation are important geological processes whose rates often require quantification. In natural settings, these processes may be taking place at a slow rate, and thus, it may not be easily visible which of these processes is occurring. Alternatively, if the effects of precipitation/dissolution are visible, it may not be clear if they are still underway or an artefact of past conditions. Moreover, these two opposing processes may flip states depending on the environmental conditions, such as, on a seasonal basis. Here, we present the technical details and preliminary results of a method using carbonate tablets and Scanning Electron Microscopy (SEM) to evaluate which process (carbonate dissolution or precipitation) is occurring, using as an example, a cave environment. Our method involves making tablets by encasing blocks of carbonate rock into resin and polishing these to form a completely flat and smooth “zero surface”. These tablets are observed under SEM in exactly the same points both before and after exposure to the field environment, using a system of marking lines at specific locations on the resin. Our results show significant differences in the before and after images of the tablet surface after just six weeks in the cave. Furthermore, the use of the insoluble resin zero surface permits a comparison of the starting height with the new dissolved/precipitated surface that can be used to quantitatively estimate the rate of dissolution/precipitation happening at a field location in a relatively short time-frame (weeks/months). This method could be used in numerous natural and industrial settings to identify these processes that can be caused purely geochemically, but also through microbialmediation and physical weathering.Raztapljanje in izločanje karbonatnih mineralov spadata med geološke procese, katerih hitrost pogosto želimo kvantitativno ovrednotiti. V naravnih okoljih so ti procesi počasni, zato običajno ni enostavno oceniti, kateri proces trenutno poteka na določeni točki, niti tega, ali so vidni učinki raztapljanja ali izločanja posledica aktivnih pogojev in procesov ali dogajanj v preteklosti. Poleg tega se nasprotujoča si procesa lahko hitro izmenjujeta zaradi sprememb v jamskem okolju, na primer sezonsko. V članku prikazujemo metodologijo in preliminarne rezultate preiskave apnenčevih ploščic (tablet), izpostavljenih pogojem raztapljanja in/ali izločanja v jamskem okolju, pod vrstičnim elektronskim mikroskopom (SEM). Metodologija obsega izdelavo testnih ploščic iz apnenca, ki smo jih zalili v epoksidno smolo, izravnali in spolirali do ravne in gladke referenčne površine. Preden smo jih izpostavili naravnim pogojem, smo ploščice označili z referenčnimi lokacijskimi točkami na meji med smolnim obodom in kamnino ter referenčna področja fotografirali pod SEM v grobovakuumskih pogojih. Nadaljne analize z SEM so pokazale znatne razlike na površini ploščic že po šestih tednih izpostavljenosti jamskim pogojem: s primerjavo z nivojem netopne epoksidne smole smo lahko neposredno ugotovili, ali je na apnenčevi ploščici prišlo do raztapljanja ali do izločanja. Referenčna mejna področja na ploščicah smo nato pod SEM analizirali v tedenskih in mesečnih intervalih z uporabo stereoskopske topografske rekonstrukcije, s katero smo kvantitativno ocenili hitrost raztapljanja oziroma izločanja karbonata. Metoda je potencialno uporabna za proučevanje geokemijskih, mikrobno induciranih in mehanskih prosesov na mikroskopski ravni v naravnih in laboratorijskih (industrijskih) pogojih

Building habit strength: A pilot intervention designed to improve food-safety behavior

The purpose of this study was to firstly design an intervention to decrease cross-contamination in the home by the development of the habitual behavior of microwaving the dishcloth/sponge and secondly to determine if this behavior could be maintained over time. Participants were randomly assigned to either a high-frequency or low-frequency reminder habit building condition or a control condition. Results indicated that for both habit building conditions, food-safety behavior significantly increased compared to the control group and these changes were maintained at follow-up. Additionally, improvement in behavior was mediated by anincrease in habit strength. The major conclusion of this study is that providing a cue to action and reminders builds food-safety habits that result in changes in food-safety behaviors. This has major implications for other food-safety interventions

Starting to smoke: a qualitative study of the experiences of Australian indigenous youth

BackgroundAdult smoking has its roots in adolescence. If individuals do not initiate smoking during this period it is unlikely they ever will. In high income countries, smoking rates among Indigenous youth are disproportionately high. However, despite a wealth of literature in other populations, there is less evidence on the determinants of smoking initiation among Indigenous youth. The aim of this study was to explore the determinants of smoking among Australian Indigenous young people with a particular emphasis on the social and cultural processes that underlie tobacco use patterns among this group. MethodsThis project was undertaken in northern Australia. We undertook group interviews with 65 participants and individual in-depth interviews with 11 youth aged 13–20 years led by trained youth ‘peer researchers.’ We also used visual methods (photo-elicitation) with individual interviewees to investigate the social context in which young people do or do not smoke. Included in the sample were a smaller number of non-Indigenous youth to explore any significant differences between ethnic groups in determinants of early smoking experiences. The theory of triadic influence, an ecological model of health behaviour, was used as an organising theory for analysis. ResultsFamily and peer influences play a central role in smoking uptake among Indigenous youth. Social influences to smoke are similar between Indigenous and non-Indigenous youth but are more pervasive (especially in the family domain) among Indigenous youth. While Indigenous youth report high levels of exposure to smoking role models and smoking socialisation practices among their family and social networks, this study provides some indication of a progressive denormalisation of smoking among some Indigenous youth. ConclusionsFuture initiatives aimed at preventing smoking uptake in this population need to focus on changing social normative beliefs around smoking, both at a population level and within young peoples’ immediate social environment. Such interventions could be effectively delivered in both the school and family environments. Specifically, health practitioners in contact with Indigenous families should be promoting smoke free homes and other anti-smoking socialisation behaviours