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.

Immelman, Wilma (2020-03)

Thesis (MMed)--Stellenbosch University, 2020.

Thesis

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.

AFRIKAANSE OPSOMMING: MALDI-TOF MS is al vir jare in gebruik in die kliniese mikrobiologie laboratoriums, maar meestal vir die identifikasie van gis en bakterieë; en tot n mindere mate vir die identifikasie van nie tuberkulose Mikobakterieë (NTM), Nocardia en skimmel. As gevolg van verskeie metodes beskikbaar vir die identifikasie van NTM, Nocardia en skimmel, die belofte van n metode wat geskik is vir al die bogenoemde organismes soos gerapporteer deur verskeie studies, sal 'n beduidende invloed hê op die werksvloei in 'n diagnostiese laboratorium. Die MALDI-TOF MS is 'n relatiewe laekoste-tegnologie met 'n vinnige omkeertyd. Beloofde resultate is in verskillende studies aangemeld en rapporteer identifikasies van 87,7% - 99.0% vir NTM, 76.0% - 98.0% vir Nocardia en 66.8% - 94.0% vir skimmels. Die doel van die studie was om die identifkasie van geselekteerde NTM, skimmel en Nocardia isolate op die MALDI-TOF-MS te vergelyk met verskeie fenotipiese en molekulêre metodes wat insluit die Genotype Mycobacterium CM / AS metodes, asook pan-bakteriële en pan-skimmel DNA volgorde benadering. Die studie sluit ook in n koste en werksvloei analise tussen die verskeie metodes. Ons studie het identifikasies van 21.8% vir NTM, 62.5% vir Nocardia en 38.5% vir skimmel geproduseer. 'n Herhalende tema vir alle organisme-identifikasies op die Vitek MS was 'n hoë mate van "geen identifikasies", ondanks die feit dat voldoende proteïen-spektrale profiele gegenereer is, sowel as die meerderheid van die organismes was verteenworrdig in die Vitek MS databasis. Ondanks beduidende probleemoplossing van die metodologie vir alle organismes, het die persentasie suksesvolle identifikasies nie verbeter nie. Die vervaardiger se verteenwoordigers kon nie die probleme gedurende hierdie studie oplos nie, wat daarop dui dat daar 'n sagteware- of hardeware verwante probleem kan wees. Op grond van die Vitek MS-instrument tekortkominge en koste- en werkvloei-analise, beveel ons die Mycobacterium CM / AS aan vir die spesifikasie van NTM's en die fenotipiese identifikasie van skimmel. Pan-Fungal-opeenvolging moet gebruik word waar die omkeertyd van kritieke belang is of waar kultuur negatiewe siektes vermoed word. Terwyl die Vitek MS 'n belofte getoon het vir Nocardia identifikasie, maak die koste daarvan, gegewe die groot stelgrootte en kort stabiliteit, die metode nie koste-effektief nie. Op dieselfde manier het die MLSA-analise die meeste identifikasies op die spesievlak verskaf, maar dit is nie koste effektief nie. Terwyl 16S rRNA-volgorde meestal slegs Nocardia op die genusvlak gerapporteer is, bly dit die enigste haalbare opsie vir bevestiging van Nocardia in die laboratorium. Samevattend benodig die Vitek MS gereelde fyninstellings en tegniese ingryping en ondersteuning. Die instrument is miskien nie geskik vir 'n laboratorium met 'n hoë deurvloei vir die identifisering van NTM's, Nocardia en skimmel sonder om die robuustheid daarvan te verbeter nie.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/108338
This item appears in the following collections: