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Prolonged heating should be avoided during the preparation of Delamanid containing Middlebrook 7H11 medium

P Rupasinghe(2) R Raeneers(2) J Vereecken(2) S Sengstake(2) L Rigouts(1,2) B C de Jong(2)

1:University of Antwerp, Wilrijk, Belgium; 2:Unit of Mycobacteriology, Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium

Prompted by the detection of false resistance, we assessed the impact of four in-vitro conditions on the minimal inhibitory concentration (MIC) of delamanid against Mycobacterium tuberculosis complex (MTBc) on Middlebrook 7H11 medium. We tested the H37Rv reference strain with two different inoculum concentrations (10⁻¹ and 10⁻² of McF 1.0 ), using polystyrene vs polypropylene labware and, using delamanid-containing media protected from light and exposed to natural light minimum for six hours. Additionally, the impact of duration to heat exposure (51°C,<40 mins vs 4-5 hours) during medium preparation before aliquoting in the tubes was tested with an extended panel of strains including H37Rv.

There was no significant impact of the type of labware used or light exposure of the drug-containing medium on the delamanid MICs for the H37Rv strain. Higher inoculum concentration (10⁻¹ of McF 1.0) didn’t affect the MICs of the H37Rv strain, yet reduced the turnaround time of the results by seven days and the occurrence of invalid results due to lack of growth on the growth controls. A systematic increase in the growth of MTBc on the media exposed to 51°C for 4-5 hours was observed compared to the media with ≤40 mins exposure, leading to false-resistant delamanid results for the H37Rv strain. Our results suggest that the length of delamanid-containing media in the water bath at 51°C should be as short as possible in order to avoid false-resistant results. Thus strict adherence to the media preparation standard operating procedures is crucial to avoid unreliable results.

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