Evolution of Mycobacterium tuberculosis complex lineages and their role in an emerging threat of multidrug resistant tuberculosis in Bamako, Mali

In recent years Bamako has been faced with an emerging threat from multidrug resistant TB (MDR-TB). Whole genome sequence analysis was performed on a subset of 76 isolates from a total of 208 isolates recovered from tuberculosis patients in Bamako, Mali between 2006 and 2012. Among the 76 patients, 61(80.3%) new cases and 15(19.7%) retreatment cases, 12 (16%) were infected by MDR-TB. The dominant lineage was the Euro-American lineage, Lineage 4. Within Lineage 4, the Cameroon genotype was the most prevalent genotype (n = 20, 26%), followed by the Ghana genotype (n = 16, 21%). A sub-clade of the Cameroon genotype, which emerged ~22 years ago was likely to be involved in community transmission. A sub-clade of the Ghana genotype that arose approximately 30 years ago was an important cause of MDR-TB in Bamako. The Ghana genotype isolates appeared more likely to be MDR than other genotypes after controlling for treatment history. We identified a clade of four related Beijing isolates that included one MDR-TB isolate. It is a major concern to find the Cameroon and Ghana genotypes involved in community transmission and MDR-TB respectively. The presence of the Beijing genotype in Bamako remains worrying, given its high transmissibility and virulence

Epidémiologie et recherche sur la tuberculose au Mali: Etat des lieux

La tuberculose reste un problème de santé publique au Mali malgré les bons scores enregistrés ces dernières années aussi bien dans la lutte que dans la recherche contre la maladie. Le nombre de cas est resté globalement stable au cours des dernières années, mais l’augmentation des cas de résistances attire de plus en plus l’attention et demande une réponse plus rigoureuse de la part des décideurs. Les recherches menées ont permis d’identifier les souches tuberculeuses circulantes au Mali, les profils de résistances et les mycobactérioses atypiques qui posent un problème de diagnostic différentiel avec la tuberculose. Les recherches ont permis aussi de mieux comprendre l’immunologie de la maladie, notamment le rôle des cellules lymphocytaires au cours de la coinfection avec le VIH et du traitement antituberculeux. Tous ces efforts doivent être renforcés pour le long terme afin de venir à bout de la maladie

Whole Genome Sequencing of <i>Mycobacterium africanum</i> Strains from Mali Provides Insights into the Mechanisms of Geographic Restriction

<div><p>Background</p><p><i>Mycobacterium africanum</i>, made up of lineages 5 and 6 within the <i>Mycobacterium tuberculosis</i> complex (MTC), causes up to half of all tuberculosis cases in West Africa, but is rarely found outside of this region. The reasons for this geographical restriction remain unknown. Possible reasons include a geographically restricted animal reservoir, a unique preference for hosts of West African ethnicity, and an inability to compete with other lineages outside of West Africa. These latter two hypotheses could be caused by loss of fitness or altered interactions with the host immune system.</p><p>Methodology/Principal Findings</p><p>We sequenced 92 MTC clinical isolates from Mali, including two lineage 5 and 24 lineage 6 strains. Our genome sequencing assembly, alignment, phylogeny and average nucleotide identity analyses enabled us to identify features that typify lineages 5 and 6 and made clear that these lineages do not constitute a distinct species within the MTC. We found that in Mali, lineage 6 and lineage 4 strains have similar levels of diversity and evolve drug resistance through similar mechanisms. In the process, we identified a putative novel streptomycin resistance mutation. In addition, we found evidence of person-to-person transmission of lineage 6 isolates and showed that lineage 6 is not enriched for mutations in virulence-associated genes.</p><p>Conclusions</p><p>This is the largest collection of lineage 5 and 6 whole genome sequences to date, and our assembly and alignment data provide valuable insights into what distinguishes these lineages from other MTC lineages. Lineages 5 and 6 do not appear to be geographically restricted due to an inability to transmit between West African hosts or to an elevated number of mutations in virulence-associated genes. However, lineage-specific mutations, such as mutations in cell wall structure, secretion systems and cofactor biosynthesis, provide alternative mechanisms that may lead to host specificity.</p></div

Patients infected with Mycobacterium africanum versus Mycobacterium tuberculosis possess distinct intestinal microbiota.

BACKGROUND:Mycobacterium tuberculosis complex (MTBC), the causative agent of tuberculosis (TB), is composed of eight subspecies. TB in West Africa, in contrast to other geographical regions, is caused by Mycobacterium africanum (MAF) in addition to M. tuberculosis (MTB), with both infections presenting similar symptoms. Nevertheless, MAF is considered to be hypovirulent in comparison with MTB and less likely to progress to active disease. In this study, we asked whether MAF and MTB infected patients possess distinct intestinal microbiomes and characterized how these microbiota communities are affected by anti-tuberculosis therapy (ATT). Additionally, we assessed if the changes in microbiota composition following infection correlate with pathogen induced alterations in host blood-gene expression. METHODS:A longitudinal, clinical study of MAF infected, MTB infected patients assessed at diagnosis and two months after start of ATT, and healthy, endemic controls was conducted to compare compositions of the fecal microbiome as determined by 16S rRNA sequencing. A blood transcriptome analysis was also performed on a subset of subjects in each group by microarray and the results cross-compared with the same individual's microbiota composition. FINDINGS:MAF participants have distinct microbiomes compared with MTB patients, displaying decreased diversity and increases in Enterobacteriaceae with respect to healthy participants not observed in the latter patient group. Interestingly, this observed elevation in Enterobacteriaceae positively correlated with enhanced inflammatory gene expression in peripheral blood and was reversed after initiation of ATT. INTERPRETATION:Our findings indicate that MAF and MTB have distinct associations with the gut microbiome that may be reflective of the differential susceptibility of West Africans to these two co-endemic infections either as biomarkers or as a contributing determinant

Screening new tuberculosis patients in Mali for rifampicin resistance at 2 months

Objective/background: The recent call for universal drug susceptibility testing (DST) for all tuberculosis (TB) patients will be difficult to meet in settings where Xpert rollout is limited, such as low prevalence of HIV and Multi-drug Resistant Tuberculosis (MDR) settings. As recommended by World Health Organization (WHO) guidelines, the success of TB treatment is measured by Ziehl–Neelsen (ZN) microscopy or auramine–rhodamine fluorescent microscopy (FM) on sputum, in which conversion to negative smear at 2 months (M) is an important predictor of treatment success, defined as a negative smear at 5M. The sputum smear that fails to convert to negative at 5M are screened for rifampicin resistance. We tested in a prospective study whether an early screen for rifampicin resistance, based on FM results at 2M, could detect MDR patients early, rather than screening all patients with GeneXpert MTB/Rif at baseline. Methods: Between February 2015 and August 2016, we enrolled new TB patients in an IRB-approved prospective cohort study at four health centers in Bamako district. Fresh sputum samples were collected at 2M and 5M to measure FM smear conversion. Patients who failed to show a decline in FM positivity at 2M (moderate or many Acid Fast Bacilli (AFB)) had their sputum tested in GeneXpert to detect rifampicin resistance. Patients who had any AFB seen at 5M were also tested using GeneXpert. Results: Of the 570 patients who were enrolled in the study, 22 (3.8%) died and 27 (4.7%) were lost to follow-up. The prevalence of HIV and TB coinfection was 12.4%, and 65.6% of the patients were male. At 2M, 32 out of 429 patients still had moderate or many AFBs in FM, and were screened by Xpert, of whom 5 (15.6%) tested rifampicin-resistant and were referred for MDR treatment. Of the 310 patients who completed 5M of treatment, 35 (11.3%) met the definition of failure (few or moderate AFB in FM) and had their sputum tested in Xpert; moreover, four (11.4%) demonstrated rifampicin resistance. In total, 67 (21.6% of 310) patients were screened by Xpert, of whom nine were detected to have MDR (or 13.4% of those screened). Conclusion: Although we cannot exclude additional MDR patients having been missed by our screening strategy, our screening algorithm at 2M detected five out of nine MDR patients. Detecting patients at 2M allowed for earlier referral, and potentially less acquired drug resistance and lower mortality. This strategy may be advantageous while awaiting further rollout of Xpert machines that will permit universal DST

Percentage of lineage-specific mutations in virulence associated genes.

<p>A) Percentage of lineage-specific mutations in coding sequences of the genes in each category. Sassetti virulence genes are genes that were identified in [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004332#pntd.0004332.ref066" target="_blank">66</a>] as being required for virulence in mice. Sassetti essential and slow growth genes were identified by Sassetti et al. under <i>in vitro</i> conditions using TraSH [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004332#pntd.0004332.ref065" target="_blank">65</a>]. Rengarajan macrophage genes were identified by Rengarajan et al. as being required for growth in macrophages [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004332#pntd.0004332.ref067" target="_blank">67</a>]. Comas antigen genes were genes identified by Comas et al. as containing T cell epitopes [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004332#pntd.0004332.ref004" target="_blank">4</a>]. The color of the bar indicates type of mutation. B) Percentage of lineage-specific pseudogenes falling into the above defined categories. Missing categories had no pseudogenes in any lineage. Lineage is indicated by the number below each bar, while ‘af’ indicates mutations found in both lineages 5 and 6 (both <i>M</i>. <i>africanum</i> lineages).</p

Average nucleotide identity (ANI) analysis indicates <i>M</i>. <i>africanum</i> and <i>M</i>. <i>tuberculosis</i> are not separate species.

<p>A) ANI values when comparing <i>M</i>. <i>africanum</i> and <i>M</i>. <i>tuberculosis</i> do not cross the ANI species threshold of 94–95%. In fact, this comparison shows that the distribution of <i>M</i>. <i>africanum</i>/<i>M</i>. <i>tuberculosis</i> comparisons (red) overlaps that of inter-lineage <i>M</i>. <i>tuberculosis</i> comparisons (purple), indicating that <i>M</i>. <i>africanum</i> should be considered another lineage of <i>M</i>. <i>tuberculosis</i>. B) Similarly, ANI values when comparing <i>M</i>. <i>bovis</i> and <i>M</i>. <i>tuberculosis</i> also overlap with inter-lineage <i>M</i>. <i>tuberculosis</i>, and indicate that <i>M</i>. <i>bovis</i> should also be considered another lineage of <i>M</i>. <i>tuberculosis</i>. C) ANI values comparing <i>M</i>. <i>africanum</i> and <i>M</i>. <i>bovis</i> (pink) also overlap inter-lineage <i>M</i>. <i>tuberculosis</i> comparisons (green).</p

Phylogenetic tree of 92 newly sequenced strains from Mali, together with 45 additional strains with whole-genome assemblies (the Assembly Collection).

<p>Nodes, lineages, and newly-sequenced Mali strains are indicated. All key nodes separating the major lineages had bootstrap values of 100%, except for the node separating <i>M</i>. <i>tuberculosis</i> lineage 1 and <i>M</i>. <i>africanum</i> lineage 5, which had a bootstrap value of 83%. Letters indicate key nodes analyzed in detail: (A) lineage 6, (B) the clade including <i>M</i>. <i>bovis</i> and lineage 6, (C) lineage 5, and (D) the clade including lineages 5, 6 and <i>M</i>. <i>bovis</i>.</p

Diversity in Mali lineage 4 and lineage 6 strains.

<p>ANI values for comparisons (A) within all Mali lineage 4 isolates and (B) within all lineage 6 isolates. Blue lines indicate mean ± standard deviation. The means of these two groups was not significantly different using the Mann-Whitney test.</p