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

Patients, specimens flow diagram and laboratory analyses.

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

Discrepancies: Spoligotype profile, lineage and sub-lineage.

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

Availability of spoligotype patterns depending on the spoligotyping method used.

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

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

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

Comparisons of cytomorphologic features with AFB positivity on direct and concentrated method of Z-N staining (N = 187).

<p>*Reactive LN = Reactive lymphadenitis.</p><p>Comparisons of cytomorphologic features with AFB positivity on direct and concentrated method of Z-N staining (N = 187).</p

Incremental yield of AFB grading on smears prepared after concentration as compared to direct smear microscopy at JUSH, Jimma, South West Ethiopia (n = 47).

<p>Incremental yield of AFB grading on smears prepared after concentration as compared to direct smear microscopy at JUSH, Jimma, South West Ethiopia (n = 47).</p

Sensitivity and specificity of direct ZN stain, concentration method and cytology against culture (n = 187).

<p>*CI = confidence interval.</p><p>Sensitivity and specificity of direct ZN stain, concentration method and cytology against culture (n = 187).</p

Correlation of cytomorphologic features of TBLN with AFB positivity (direct &concentrated smear) and culture positivity (N = 82).

<p>Correlation of cytomorphologic features of TBLN with AFB positivity (direct &concentrated smear) and culture positivity (N = 82).</p