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Deeplex Myc/TB directly on sputum detects more mixed infections and heteroresistance compared to culture-based whole genome sequencing.

F Massou(1,2,3) P Supply(4) W Mulders(2) B C de Jong(2) D Affolabi(1) L Rigouts(2,3) C J Meehan(5)

1:Laboratoire de Reference des Mycobacteries; 2:Institute of Tropical Medicine; 3:University of Antwerp; 4:Genoscreen; 5:Nottingham Trent University

Whole genome sequencing (WGS) analysis of M. tuberculosis (MTB) still relies on culture, which may introduce bias by losing fastidious growers and/or minority variants. Direct sequencing on sputum may provide more clinically and epidemiologically relevant information. 

In the DIAMA study comprising 9 sub-Saharan African countries, we enrolled equal numbers of Xpert positive patients with rifampicin resistant (RR)-TB and retreatment patients with RS-TB. We compared the performance of Deeplex Myc/TB (Genoscreen, France) directly on sputum with WGS from respective positive cultures, to detect the presence of heteroresistance (resistance mutations < 90%) and mixed infections. Analysis was done using an integrated platform (Deeplex) and TB profiler Vs4.4.2 (WGS).

Among a total of 2132 samples, 1814 yielded valid results by Deeplex and 1131 by culture followed by WGS (cWGS). Multiple lineages were detected in 10% of specimens by Deeplex versus 1% of isolates by cWGS .This difference was primarily driven by Lineages 5 and 6  not being detected in mixed infections by Cwgs (OR, p< 0.01). Single clone heteroresistant sub-populations were detected in 9% by Deeplex and 6% by cWGS.

These findings suggest that culture-based WGS misses large proportions of mixed infections and heteroresistance, likely due to inherent biases selecting specific strains during the culturing process. Strains missed by cWGS were primarily L5 and L6, which has large implications for cWGS-based epidemiological studies in West Africa. Further work is required to develop direct from clinical sample WGS to overcome these biases in future studies.

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