Novel approaches in bacterial keratitis

Bacterial keratitis is a serious ophthalmic disease with significant visual morbidity. The aims of this thesis are to investigate (1) the prevalence and type of bacterial virulence factors in the two major species causing keratitis; Staphylococcal aureus and Pseudomonas aeruginosa and (2) the pharmacodynamics and pharmacokinetics of meropenem, a potentially novel antimicrobial for use in keratitis. The prevalence, genetic diversity and clinical relevance of the lukSF-PV gene, encoding the bacterial toxin Panton Valentine Leukocidin, were investigated in S. aureus, isolated from cases of bacterial keratitis in the UK. Patients with lukSF-PV+ve S. aureus were found to be associated with a trend to worse clinical outcome and more surgical interventions, with an effect unrelated to minimum inhibitory concentrations (MICs). This suggests that lukSF-PV may be an important virulence factor in S. aureus associated keratitis. The genetic characteristics of P. aeruginosa isolates collected from patients with bacterial keratitis were compared between two time periods; 2003-04 and 2009-10 using an Array Tube genotyping system. 71% of keratitis-associated P. aeruginosa isolates clustered together, with no evidence for major variations in the distribution of clone types between the two time periods. The “core keratitis cluster” was found to be related to the P. aeruginosa eccB clonal complex, which is associated with adaptation to survival in environmental water. This suggests that adaptation to environmental water is a key factor in the ability of P. aeruginosa to cause keratitis. Further analysis of clinical data and studies involving additional sets of patients for verification of this hypothesis will provide a clearer picture. Pharmacodynamic properties of commonly used and potentially novel antimicrobials were determined by calculating MICs against isolates from patients with keratitis. Antimicrobial combinations were investigated for synergy or antagonism against isolates of S. aureus and P. aeruginosa using E-Tests. Meropenem was identified as a potentially novel antimicrobial for use in keratitis, offering broad-spectrum cover against both Gram-positive and -negative microorganisms. Meropenem was also shown to show synergistic properties when used in combination with other commonly used antimicrobials. In light of these results, the antimicrobial meropenem was chosen to undergo further pharmacokinetic studies. MTT and Live Dead toxicity assays were performed on human keratocytes and human corneal epithelial cells treated with meropenem. Meropenem was found to have low toxicity against these cells. Corneal penetration of meropenem was assessed in human cadaver corneo-scleral discs mounted onto artificial anterior chambers. The concentration of meropenem in the aqueous estimated by HPLC and disc diffusion bioassay exceeded the MIC90 of E. coli in all 18 corneas tested after the 45 minutes sample point

Contribution of a Real Depth Distance Stereoacuity Test to Clinical Management

Measurement of Stereopsis forms an important part of the clinical assessment of patients with disorders of ocular motility. The introduction of a real depth distance stereoacuity test (FD2) was evaluated in clinical practice and to what extent the introduction affected clinical management. Seventy-three patients under evaluation before and following the introduction of the test were included. Combined thresholds were measured at near using the Frisby and TNO test and at distance using the FD2. Fifty healthy controls were included. Forty-five patients demonstrated Stereopsis using the FD2 and 23 of these had a change in their management based in part on their responses using the FD2. Patients with evidence of Stereopsis using the FD2 were significantly more likely to have change in their management than expected from the whole sample (P = .02). The introduction of a real depth distance stereoacuity test into clinical practice contributed to a change in management when used in conjunction with other tests. The usefulness of the FD2 is limited by its range at 6 m. Use at closer distances necessitates the calculation of binocular threshold from the combined and monocular threshold

Aspectos teórico-numéricos dos métodos SPH e MPS

Currently, due to the technological advances the use of particle methods is gaining ground in the simulations of fluid flow. The first particle method to be developed was the Smoothed Particle Hydrodynamics (SPH) that was very efficient for compressible flow problems, but inefficient for incompressible ones. Thus, there was some strategy to solve incompressible flow problems as the incompressible Smoothed Particle Hydrodynamics (ISPH) and the Moving Particle Semi-Implicit (MPS); in both methods the pressure is updated by a Poisson equation. For an approximation of the Navier-Stokes equations it is first needed a good approximation for the Poisson equation. This paper discusses the following particles methods: Smoothed Particle Hydrodynamics (SPH) and Moving Particle Semi-Implicit (MPS). The discretization of differential operators by these methods is done through the approximation of the kernel and also by particles. A comparative study of different discretizations were made. In order to know if the parameters used in the literature for the SPH and MPS methods provide a good solution for Poisson equation, have been performed several tests by varying the parameters with and without the borders treatment. This work also proposed a strategy to solve the oscillation problem in advection equation with discontinuity in the initial conditions and the results were very satisfactoryAtualmente, devido ao grande avanço tecnológico o uso dos métodos de partículas vêm ganhando espaço nas simulações de escoamento de fluido. O primeiro método de partículas a ser desenvolvido foi o Smoothed Particle Hydrodynamics (SPH) que se mostrou bastante eficiente para problemas de escoamento compressível, mas ineficiente para escoamento incompressível. Desta forma, surgiu algumas estratégias para resolver problemas de escoamento incompressível como o Incompressible Smoothed Particle Hydrodynamics (ISPH) e o Moving Particle Semi-Implicit (MPS); em ambos os métodos a pressão é atualizada por um equação de Poisson. Portanto para obter uma boa aproximação das equações de Navier-Stokes é necessário antes ter uma boa aproximação da equação de Poisson. Neste trabalho são abordados os métodos de partículas Smoothed Particle Hydrodynamics (SPH) e Moving Particle Semi-Implicit (MPS). A discretização dos operadores diferenciais por esses métodos é feita por meio da aproximação do núcleo e também por partículas. Um estudo comparativo entre discretização foram efetuadas. Afim de saber se os parâmetros utilizados na literatura dos métodos de partículas SPH e MPS dão uma boa solução para equação de Poisson foram realizados vários testes variando os parâmetros com e sem o tratamento de fronteira. Neste trabalho também foi proposta uma estratégia para resolver o problema de oscilação na solução da equação de advecção com descontinuidade nas condições iniciais e os resultados foram bem satisfatórioCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Numerical study of transitional flow over concave surface with non stationary disturbances

A geração dos vórtices de Görtler é o primeiro estágio do processo de transição laminar turbulento em um escoamento de camada limite sobre uma superfície côncava. Estes vórtices geram fortes distorções tanto no perfil de velocidade quanto no perfil de temperatura intensificando a taxa de transferência de calor e o coeficiente de arrasto em relação ao escoamento laminar. A fim de maximizar ou minimizar os efeitos dessas duas propriedades neste tipo de escoamento, há vários estudos com o intuito de entender os mecanismos da formação dos vórtices de Görtler para poder retardar ou antecipar a geração deles. Este trabalho investiga a evolução, a estrutura e a influência das perturbações não estacionárias em um escoamento de camada limite sobre uma superfície côncava através da simulação numérica com métodos de alta ordem de precisão. Os resultados mostram que quando o comprimento de onda transversal e a frequência temporal aumentam, o modo subharmônico estacionário (0,2) domina o escoamento gerando os vórtices de Görtler estacionários, caso contrário, o modo fundamental (1,1) domina o escoamento e os vórtices não estacionários são gerados. As perturbações não estacionárias retardam o surgimento os vórtices de Görtler, pois a taxa de crescimento do modo fundamental (1,1) diminui quando a frequência aumenta independentemente do comprimento de onda transversal. A taxa de transferência de calor e o coeficiente de arrasto são menores para os vórtices não estacionários do que para os vórtices estacionários.The generation of Görtler vortices is the first stage of the turbulent laminar transition process in a boundary layer flow over a concave surface. These vortices produce distortions both in the velocity profile and in the temperature profile, intensifying the heat transfer rate and the drag coefficient compared to the laminar flow. There are several studies to understand the mechanisms of the formation of Görtler vortices to delay or anticipate their generation to maximize or minimize the effects of these two properties in this type of flow. This work investigates the evolution, structure, and influence of non-stationary disturbances in a boundary layer flow over a concave surface through numerical simulation with high-precision methods. The results show the stationary sub-harmonic mode (0,2) dominates the flow generating the stationary Görtler vortices as the spanwise wavelength and time-frequency increase, otherwise, the fundamental mode (1,1) dominates the flow and generated the unsteady vortices. Non-stationary disturbances delay the appearance of Görtler vortices since the growth rate of the fundamental mode (1,1) decreases when the frequency increases regardless of the transverse wavelength. The heat transfer rate and the drag coefficient for the unsteady vortices are lower than for the steady vortices

Impression membrane for the diagnosis of microbial keratitis

Purpose: To evaluate a corneal impression membrane (CIM) for isolation of bacteria, fungi and acanthamoeba in suspected microbial keratitis. Methods: Consecutive patients presenting with suspected microbial keratitis were included. For each patient, samples were collected in a random order using a surgical blade and a 4-mm-diameter polytetrafluoroethylene CIM disc, and transported in brain heart infusion broth. Risk factors, best corrected visual acuity (BCVA), size, location, depth and healing time of the ulcer were recorded. The microbial isolation rate was used to compare sampling methods. Results: 130 patients were included (mean age 62.6 years, SD 19.0). An antimicrobial had been used prior to presentation in 36 (27.7%) patients. Mean major and minor ulcer diameters were 2.1 mm (SD 2.0) and 1.6 mm (SD 1.7). Mean healing time was 12.4 days (SD 13.6). BCVA at presentation and following healing was 0.7 (SD 0.7) and 0.62 (SD 0.7) (p=0.34). There were 66 isolates (50.8%); 53 (40.8%) using a CIM and 35 (26.9%) using a blade (p=0.02). Staphylococcus aureus and coagulase-negative staphylococci were the commonest isolates. Isolation rate was not influenced by organism type, although in four cases Acanthamoeba spp. were isolated; three using CIM and one a blade. Conclusions: In this study, the isolation of microorganisms from cases of suspected microbial keratitis was significantly higher using a CIM than a surgical blade. A CIM may be a useful alternative or addition for sample collection in microbial keratitis

Recurrent bacterial keratitis

Purpose.: To investigate the rate of recurrent bacterial keratitis, associated bacteria, and surgical intervention.Methods.: Patients with suspected bacterial keratitis were identified from microbiological requests over a 16-year period between 1995 and 2010. Recurrences and number of surgical interventions were analyzed according to bacterial type.Results.: A total of 2418 patients were included, of whom 2124 (87.84%) had only one episode of keratitis, 294 (12.15%) at least two, 88 (3.63%) at least three, 40 (1.65%) at least four, and 22 (0.91%) five or more episodes. The bacterial isolation rate was 35.74% (SD 9.41%), increasing to 56.01% in patients with two or more episodes. There was an increase in the isolation of Staphylococcus aureus with increasing number of episodes (P = 0.008), and S. aureus occurred more commonly in patients with recurrent disease due to the same bacterial group (P = 0.04). Patients whose recurrent keratitis was associated with S. aureus had a higher rate of requiring subsequent corneal transplantation (7 of 10) compared to those with Enterobacteriaceae (2 of 7), Pseudomonas aeruginosa (2 of 4), streptococci (2 of 5), or coagulase-negative staphylococci (none of 8) (P = 0.02).Conclusions.: S. aureus is particularly associated with recurrent keratitis. Identification and treatment of the possible source of the infection may be necessary to reduce the risk of recurrent disease. The potential for the autocthonous S. aureus colonizing the nasopharynx or conjunctiva or lid margin to be a reservoir for recurrent keratitis suggests that decolonization of S. aureus could be considered as a potential intervention in those patients with recurrent disease