Medical Microbiology

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Browsing Medical Microbiology by browse.metadata.advisor "Hoek, Dr Kim"

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    Optimisation and comparison of a phenotypic maldi-tof assay with molecular and phenotypic methods for the rapid identification of selected fungal, nocardia and nontuberculous mycobacteria.
    (Stellenbosch : Stellenbosch University, 2020-03) Immelman, Wilma; Hoek, Dr Kim; Wasserman, Elizabeth; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Microbiology.
    ENGLISH ABSTRACT: Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been utilised in clinical microbiology laboratories for several years, but is mostly used for the rapid and accurate identification of bacteria and yeasts; and to a lesser extent for nontuberculous mycobacteria (NTM), Nocardia and moulds. Due to the variety of methods used for the identification of NTM, Nocardia and moulds , the promise of an identification method ‘fit for all’, as reported in some studies, would have a significant impact on the work flow in a diagnostic laboratory. The MALDI-TOF MS is a relatively low-cost technology with a quick turnaround time following culture. Promising results were reported in various studies and includes identification rates of 87.7% - 99.0% for NTM, 76.0% - 98.0% for Nocardia and 66.8% - 94.0% for moulds. The aim of this study was to compare the identification of selected NTM, Nocardia and moulds using MALDI-TOF MS with various phenotypic and molecular methods including routine fungal culture, the Genotype Mycobacterium CM / AS assays, as well as a pan-bacterial and pan-fungal sequencing approach. The study also included a cost and workflow analysis between the different methods employed. Our study produced identification rates of 21.8% for NTM, 62.5% for Nocardia and 38.5% for moulds. A recurring theme for all organism identifications on the Vitek MS was a high rate of “no identifications”, despite adequate protein spectral profiles being generated as well as the majority of the organisms being represented in the Vitek MS Knowledge Base Database. Despite significant troubleshooting of the methodology for all organisms, the percentage of successful identifications did not improve. The manufacturer representatives were unable to resolve the issues during the course of this study, suggesting that there may be a software or hardware related problem. Based on the Vitek MS instrument shortcomings and cost and workflow analysis, we recommend the Mycobacterium CM/AS kit for the speciation of NTMs and the phenotypic identification of moulds. ITS Pan-Fungal sequencing should be used where turnaround time is critical or where culture negative disease is suspected. While the Vitek MS showed promise for Nocardia identification, the cost thereof given the large kit size and short stability, makes cost prohibitive. Similarly MLSA analysis provided the most identifications to the species level, but is cost prohibitive. While 16S rRNA sequencing mostly only reported Nocardia to the genus level, it remains the only feasible option for Nocardia confirmation in the laboratory. In summary, the Vitek MS requires regular fine-tuning and technical intervention and support. The instrument is perhaps not suited to a high throughput laboratory for the identification of NTMs, Nocardia and moulds without increasing it’s robustness.