Improving the laboratory diagnosis of tuberculosis

: a self-contained category 3 laboratory

Abstract

The laboratory detection of tuberculosis using culture based methods represents the current gold standard however it retains a number of weaknesses. There is a lack of understanding of how culture results relate to clinical outcomes. Additionally, as culturing tuberculosis is hazardous it necessitates a biosafety level 3 laboratory, these are expensive and therefore limited in availability. Finally, as M. tuberculosis is a slow growing organism culture results take up to 6-weeks. This thesis set out to construct a self-contained category 3 laboratory that could rapidly detect tuberculosis and along the way an investigation in to how the laboratory detection of tuberculosis relates to clinical outcomes was carried out.


The decontamination process was shown to have a greater detrimental effect on actively growing mycobacteria compared to non-replicating mycobacteria when using an artificial sputum model. It was concluded that the decontamination process has an impact on how treatment response is monitored with actively growing cells being underrepresented.


A technique was developed to measure the growth rate of mycobacteria during the routine processing of samples. When measuring the growth rates of clinical isolates, it was observed that a greater bacterial load correlated with a faster growth rate in two mycobacterial species. With bacterial load being prognostic of clinical outcomes it identifies for the first time that a quick growth rate could be driving a patients bacterial load with important implications for prognosis.

Moreover, the growth rate was determined to be influential in the time to positivity however the size of this effect was influenced by the inoculum size. The development of an easy technique to measure the growth rate of routine clinical samples developed here will facilitate large studies on mycobacterial fitness.

A self-contained laboratory in a cartridge format (cat 3 cartridge) was designed and constructed using rapid prototyping techniques. Using this devices it was possible to decontaminate a sample, incubate the culture and get the same results as a conventional technique. This was all done using a low cost design that inherently provides biosafety protection This is a tool that could become a self-contained category 3 laboratory, facilitating an expansion of culture based diagnostic services that is not possible with previous technology.


Two techniques were evaluated to rapidly detect growth, determine resistance and detect growth rates. These techniques were shown to be comparable with the current best culture techniques for the detection of growth. These were also developed to function as a drug sensitivity assay while also measuring the growth rate.


Future work for combining the cat 3 cartridge with either of the rapid detection techniques developed here would create an inexpensive system that is widely available. The combination could facilitate an increase in case detection rate and a reduction in the time to diagnosis. Finally with the ability to quantify growth rates it could provide crucial information on an individual isolate.

Date of Award	23 Jun 2017
Original language	English
Awarding Institution	University of St Andrews
Supervisor	Stephen Henry Gillespie (Supervisor)