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

Abstract

BackgroundThis 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 findingsSmear-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 Löwenstein 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/ SignificanceAncestral 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.Trial registrationClinicalTrials.gov NCT02744469

Mycobacterium tuberculosiscomplex lineage 5 exhibits high levels of within-lineage genomic diversity and differing gene content compared to the type strain H37Rv

“…The absence of these genes in most L5 isolates suggests that L5 strains would be less likely to survive in macrophages (Rv1978), have reduced growth in vitro (Rv1994c , as previously found by Sanoussi et al (Sanoussi et al 2017)), be less immune-evasive, less persistent and less virulent (Rv2074) than L4 strains (at least H37Rv; as previously suggested for L5 and L6 strains (Coscolla and Gagneux 2014;Reiling et al 2013)). In addition, the absence of Rv2074 in most L5 (and in the complete genomes of L6 and M. bovis as well) suggests that L6 and M. bovis strains would also be less likely to be immune-evasive, to be persistent and to survive than their L4 counterparts.…”

“…Several phenotypic and genotypic features separate L5 and L6 from the other human-adapted lineages. Some TB diagnostics have a lower performance for L5 and L6 strains, compared to other MTBC lineages (Sanoussi et al 2018;Ofori-Anyinam et al 2016) and these lineages are less likely to grow in culture (Sanoussi et al 2017), with dysgonic appearance on solid medium (Leao et al 2004;Pattyn et al 1970;Sanoussi et al 2018;Niemann et al 2004). Mutations in genes essential for growth in culture were identified for L6 strains (Gehre et al 2013), yet for L5 strains the reasons for the difficulty in growth remain unclear but may be related to the presence of the TbD1 region and the absence of RD9 (Brosch et al 2002).…”

Comparative Genomics Shows Differences in the Electron Transport and Carbon Metabolic Pathways ofMycobacterium africanumrelative toMycobacterium tuberculosisand suggests an adaptation to low oxygen tension

“…Overall, more convergence occurred between MTB L1, MTB L3 and the MAF lineages. This is interesting as the MAF lineages, MTB L1 and L3 lineages all reportedly grow relatively slower than the MTB L2 and L4 lineages (14, 65, 83, 84, 107). We postulate that the slower growth rate of these lineages and the convergence we observe in their metabolic pathways, may be linked to their similar migration and dispersal patterns in Africa and Eurasia (108), where L2 and L4 have become widely dispersed, while L5, L6, and L7, had more geographically restricted expansion, adapting to more specific hosts.…”

“…The only potentially deleterious mutations we detected in the Succinate dehydrogenases were in sdhA of SDH2 in a clade of MAF L6 and in SDH1, Rv0249c , in a clade of MTB L1 (Figure 5 and Figure 9). Given the essential role of SDH1 for growth and survival, where the deletion of SDH1 was shown to impair the rate of respiration through the Electron Transport Chain and to reduce cell viability (87), future studies should determine if electron transport and aerobic respiration are affected in certain MTB L1 strains, as recent studies report slower growth and a lower odds to grow in culture for MTB L1 and the MAF lineages (65, 83).…”

“…This convergence largely found in MTB lineages 1, 2, 3 and the MAF lineages, indicates there may be a selective advantage for this. Interestingly, these lineages, compared to MTB L4 strains, have been associated with slower growth and cytokine induction patterns suggestive of immune evasion (8, 14, 65, 83–85, 107, 110, 111).…”