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Pathogens of the Mycobacterium tuberculosis complex (MTBC) are considered to be monomorphic, with little gene content variation between strains. Nevertheless, several genotypic and phenotypic factors separate strains of the different MTBC lineages (L), especially L5 and L6 (traditionally termed Mycobacterium africanum ) strains, from each other. However, this genome variability and gene content, especially of L5 strains, has not been fully explored and may be important for pathobiology and current approaches for genomic analysis of MTBC strains, including transmission studies. By comparing the genomes of 355 L5 clinical strains (including 3 complete genomes and 352 Illumina whole-genome sequenced isolates) to each other and to H37Rv, we identified multiple genes that were differentially present or absent between H37Rv and L5 strains. Additionally, considerable gene content variability was found across L5 strains, including a split in the L5.3 sub-lineage into L5.3.1 and L5.3.2. These gene content differences had a small knock-on effect on transmission cluster estimation, with clustering rates influenced by the selected reference genome, and with potential overestimation of recent transmission when using H37Rv as the reference genome. We conclude that full capture of the gene diversity, especially high-resolution outbreak analysis, requires a variation of the single H37Rv-centric reference genome mapping approach currently used in most whole-genome sequencing data analysis pipelines. Moreover, the high within-lineage gene content variability suggests that the pan-genome of M. tuberculosis is at least several kilobases larger than previously thought, implying that a concatenated or reference-free genome assembly ( de novo ) approach may be needed for particular questions.

Human tuberculosis (TB) is caused by members of the Mycobacterium tuberculosis complex (MTBC). The MTBC comprises several human-adapted lineages known as M. tuberculosis sensu stricto , as well as two lineages (L5 and L6) traditionally referred to as Mycobacterium africanum . Strains of L5 and L6 are largely limited to West Africa for reasons unknown, and little is known of their genomic diversity, phylogeography and evolution. Here, we analysed the genomes of 350 L5 and 320 L6 strains, isolated from patients from 21 African countries, plus 5 related genomes that had not been classified into any of the known MTBC lineages. Our population genomic and phylogeographical analyses showed that the unclassified genomes belonged to a new group that we propose to name MTBC lineage 9 (L9). While the most likely ancestral distribution of L9 was predicted to be East Africa, the most likely ancestral distribution for both L5 and L6 was the Eastern part of West Africa. Moreover, we found important differences between L5 and L6 strains with respect to their phylogeographical substructure and genetic diversity. Finally, we could not confirm the previous association of drug-resistance markers with lineage and sublineages. Instead, our results indicate that the association of drug resistance with lineage is most likely driven by sample bias or geography. In conclusion, our study sheds new light onto the genomic diversity and evolutionary history of M. africanum , and highlights the need to consider the particularities of each MTBC lineage for understanding the ecology and epidemiology of TB in Africa and globally.

Purpose: Differentiation of the Mycobacterium tuberculosis complex (MTBc) from non-tuberculous mycobacteria (NTM) is important for tuberculosis diagnosis and is a prerequisite for reliable phenotypic drug-resistance testing. We evaluated the performance of the rapid MPT64 antigen identification test for the detection of Mycobacterium africanum lineage 5 (MAF L5)., Methodology: Smear-positive tuberculosis patients' sputa were included prospectively. Culture was performed on Löwenstein-Jensen medium and, when positive, the MPT64 test and the classical para-nitro benzoic acid susceptibility and heat-labile catalase (PNB/catalase) identification tests were performed. The MPT64 test was repeated 14 days after an initially negative first testing. Direct spoligotyping was performed for MTBc lineage determination., Results: In total, 333 isolates were tested for all of the methods. Three hundred and twenty-two (96.7 %) were pure MTBc, by agreement between spoligotyping and PNB/catalase, and 11 were NTM or a mixture of MTBc/NTM. The MPT64 test conducted on day zero of culture-positivity correctly identified most of the pure MTBc isolates (93.2 %, 300/322), but it failed to detect 24 % of the L5 isolates (18/75) versus 2 % (4/202) of the L4 ones [OR=15.6 (5.3-45.8), P<0.0001], with improved sensitivity for L5 detection on repeat testing after 14 days. The L5-wide non-synonymous single-nucleotide polymorphism in the mpt64 gene may explain the poor performance of the MPT64 test for L5., Conclusion: The MPT64 test has a lower sensitivity for detecting L5 isolates of the MTBc, and can be considered as a first-screening test that should be confirmed by another identification method when it produces negative results in countries with L5. Given the microbiological bias in both the isolation and identification of MAF lineages, diagnostics with high sensitivity for direct testing on clinical material are preferable.

Purpose. The aim was to assess the performance of both cetylpyridinium chloride (CPC) and OMNIgene•SPUTUM (OMNI) reagents for the maintenance of Mycobacterium tuberculosis viability in sputum prior to recovery by culture. Methodology. Using 312 sputa, we evaluated the performance of the two reagents using culture on Löwenstein-Jensen medium after sputum storage in CPC or OMNI for up to 28 days. In addition, the viability of M. tuberculosis isolates stored in both reagents was assessed. Results. The contamination rates for freshly processed samples and those stored in CPC were not statistically different, while the contamination rate for OMNI was significantly lower than that for fresh sputa ( P =0.026 for 8 days and P =0.002 for 28 days of storage). The culture positivity for fresh sputa (81.7 %) was similar to that for samples stored in CPC, regardless of the storage time (89.8 % for CPC-8 and 73.0 % for CPC-28). For OMNI-preserved samples, the culture positivity was similar after 8 days of storage (84.2 %), but decreased significantly after 28 days (42.7 %; P <0.0001) compared to fresh sputa, CPC-8, CPC-28 and OMNI-8. There was a significant loss of viability for the H37Rv strain when it was stored in OMNI at room temperature beyond 8 days compared to CPC, but storage at 37 °C decreased recovery from both CPC- and OMNI-stored suspensions. Conclusion. Culture from sputum stored for 8 days at room temperature in OMNI or CPC gave comparable culture positivity rates to culture from fresh sputum, but after 28 days of storage the performance of OMNI decreased significantly compared to CPC.

Purpose: Rapid and inexpensive tests for detecting extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae are needed, particularly in low-resource countries where infections with these bacteria constitute a major public health issue. The recently described ESBL NDP test performed well in developed countries. This study was designed to assess performance, cost and feasibility of this test in positive blood cultures, in Cotonou, Benin (West Africa)., Methodology: The test was performed on 175 positive Bactec broth blood cultures containing Enterobacteriaceae, and blindly compared with the double-disc synergy test (DDST) for the phenotypic detection of ESBL producers., Results: There was a complete agreement between the ESBL NDP test and the DDST. On average, the time to give results was 37 min for a sample and the cost was US$ 7.3., Conclusion: The ESBL NDP test is rapid, relatively affordable and performed well in our setting.

We have evaluated two simple, rapid and low-cost colorimetric methods for the detection of multidrug-resistant Mycobacterium tuberculosis. A total of 151 M. tuberculosis strains were tested for resistance to rifampicin (RMP) and isoniazid by resazurin microplate assay (REMA) and nitrate reductase assay (NRA) in comparison with the conventional proportion method (PM) on Löwenstein-Jensen medium. A complete agreement was found between NRA and PM, while one false RMP-susceptible result was found by REMA. REMA and NRA tests are rapid and inexpensive, and could be good alternatives to the conventional PM in low-resource countries.