Genotypic characterization directly applied to sputum improves the detection of Mycobacterium africanum West African 1, under-represented in positive cultures

Background

This study aimed to compare the prevalence of Mycobacterium tuberculosis complex (MTBc) lineages between direct genotyping (on sputum) and indirect genotyping (on culture), to characterize potential culture bias against difficult growers.

Methodology/Principal findings

Smear-positive sputa from consecutive new tuberculosis patients diagnosed in Cotonou, (Benin) were included, before patients had started treatment. An aliquot of decontaminated sputum was used for direct spoligotyping, and another aliquot was cultured on Lowenstein Jensen (LJ) medium (90 days), for indirect spoligotyping. After DNA extraction, spoligotyping was done according to the standard method for all specimens, and patterns obtained from sputa were compared versus those from the derived culture isolates. From 199 patient's sputa, 146 (73.4%) yielded a positive culture. In total, direct spoligotyping yielded a pattern in 98.5% (196/199) of the specimens, versus 73.4% (146/199) for indirect spoligotyping on cultures. There was good agreement between sputum-and isolate derived patterns: 94.4% (135/143) at spoligotype level and 96.5% (138/143) at (sub) lineage level. Two of the 8 pairs with discrepant pattern were suggestive of mixed infection in sputum. Ancestral lineages (Lineage 1, and M. africanum Lineages 5 and 6) were less likely to grow in culture (OR = 0.30, 95% CI (0.14 to 0.64), p = 0.0016); especially Lineage 5 (OR = 0.37 95% CI (0.17 to 0.79), p = 0.010). Among modern lineages, Lineage 4 was over-represented in positive-culture specimens (OR = 3.01, 95% CI (1.4 to 6.51), p = 0.005).

Conclusions/Significance

Ancestral lineages, especially M. africanum West African 1 (Lineage 5), are less likely to grow in culture relative to modern lineages, especially M. tuberculosis Euro-American (Lineage 4). Direct spoligotyping on smear positive sputum is effective and efficient compared to indirect spoligotyping of cultures. It allows for a more accurate unbiased determination of the population structure of the M. tuberculosis complex.