PCR Techniques of Dried Sputum Using a Spotcard

Overview

Molecular testing for mutations in M. tuberculosis genes associated to resistance of anti tuberculosis (TB) drugs is already part of standard laboratory TB diagnostic. This implicates earlier knowledge of possible resistance and thus prevents unnecessary treatment and the chance of treatment failure or treatment related toxicity. The molecular laboratory diagnostics is widely spread in high income, low TB endemic countries. However, the low income countries lack widespread facilities to test for susceptibility, either genotypic or phenotypic. Performing molecular diagnostics on sputum collected with a spot card could improve accessibility to molecular testing. This study examines if sputum collected and put on spot cards could be used for multiple molecular tests for the detection, identification and susceptibility prediction of TB. This means that DNA extraction of the sputum from the spot card should be feasible. The study is a pilot study with adult patients of the tuberculosis department of University Medical Centre Groningen (UMCG) Beatrixoord, Haren as subjects. The sputum produced will be collected, dried on spot cards, and DNA extraction from the card will be tested. If molecular detection is positive for the tuberculosis bacteria additional tests will be performed. Based on the present/absent of mutations in the genes associated to resistance susceptibility can be preditec, different molecular techniques will be performed to identify possible mutations. Furthermore, sputum will be collected as patients produce so. Sputum samples with low bacterial load can be tested as well and can test the sensitivity of the procedure. Lastly, techniques like RNA detection will be tested to identify the bacterial load. This can be done if more than one sample of patients were collected. Subjects will be selected on age, participation in standard TDM and drug use. Demographic parameters will be analysed. Sputum samples will be taken twice a week (on Tuesday and Friday).

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