Time to culture positivity (on LJ medium) across lineages.

<p>Time to culture positivity (on LJ medium) across lineages.</p

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

<div><p>Background</p><p>This study aimed to compare the prevalence of <i>Mycobacterium tuberculosis</i> complex (MTBc) lineages between direct genotyping (on sputum) and indirect genotyping (on culture), to characterize potential culture bias against difficult growers.</p><p>Methodology/Principal findings</p><p>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 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 <i>M</i>. <i>africanum</i> 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).</p><p>Conclusions/ Significance</p><p>Ancestral lineages, especially <i>M</i>. <i>africanum</i> West African 1 (Lineage 5), are less likely to grow in culture relative to modern lineages, especially <i>M</i>. <i>tuberculosis</i> 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 <i>M</i>. <i>tuberculosis</i> complex.</p><p>Trial registration</p><p>ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT02744469" target="_blank">NCT02744469</a></p></div

Availability of spoligotype patterns depending on the spoligotyping method used.

<p>Availability of spoligotype patterns depending on the spoligotyping method used.</p

Discrepancies: Spoligotype profile, lineage and sub-lineage.

<p>Discrepancies: Spoligotype profile, lineage and sub-lineage.</p

Patients, specimens flow diagram and laboratory analyses.

<p>Patients, specimens flow diagram and laboratory analyses.</p

Effect of prior culture on spoligotyping analysis for MTBc lineage detection.

<p>Effect of prior culture on spoligotyping analysis for MTBc lineage detection.</p

Additional file 1: of Follow-up and tracing of tuberculosis patients who fail to attend their scheduled appointments in Cotonou, Benin: a retrospective cohort study

STROBE Statement—Checklist of items that should be included in reports ofcohort studies. (PDF 18 kb

Population structure of <i>M. africanum</i> West Africa 1 (MAF1).

<p>A UPGMA tree was constructed including 141 MAF1 isolates from Benin, Sierra Leone and Nigeria. Ten sub-lineages were identified. Seven isolates could not be assigned to any of the defined sub-lineages.</p

Geographical distribution of identified <i>M. africanum</i> West Africa 1 (MAF1) sub-lineages.

<p>(CRS = Cross River State).</p

Geographical location of various study sites in Benin and Nigeria and the prevalence of <i>M. africanum</i> West Africa 1 (MAF1) and West Africa 2 (MAF2).

<p>Data from Benin was generated in the present study, the prevalence in Ibadan, Abuja and Nnewi was extracted and estimated from Lawson <i>et al.. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077000#pone.0077000-Lawson1" target="_blank">[9]</a>, whereas the prevalence in the Cross River State derived from Thumamo <i>et al.. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077000#pone.0077000-Thumamo1" target="_blank">[10]</a>.</p