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

C N'Dira Sanoussi; Dissou Affolabi; Leen Rigouts; Séverin Anagonou; Bouke de Jong

doi:10.1371/journal.pntd.0005900

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

PLoS Negl Trop Dis. 2017 Sep 1;11(9):e0005900. doi: 10.1371/journal.pntd.0005900. eCollection 2017 Sep.

Authors

C N'Dira Sanoussi^{1

2

3}, Dissou Affolabi², Leen Rigouts^{1

3}, Séverin Anagonou², Bouke de Jong¹

Affiliations

¹ Mycobacteriology Unit, Institute of Tropical Medicine, Antwerp, Belgium.
² Laboratoire de Référence des Mycobactéries, Centre National Hospitalier Universitaire de Pneumo-Phtisiologie de Cotonou, National Tuberculosis Programme, Cotonou, Benin.
³ Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium.

Abstract

Background: This 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 findings: 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 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/ significance: Ancestral 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 registration: ClinicalTrials.gov NCT02744469.

Publication types

Multicenter Study
Observational Study

MeSH terms

Adolescent
Adult
Aged
Aged, 80 and over
Bacterial Typing Techniques
Benin / epidemiology
DNA, Bacterial / genetics
Female
Genotype
Humans
Male
Middle Aged
Mycobacterium tuberculosis / classification
Mycobacterium tuberculosis / genetics*
Mycobacterium tuberculosis / isolation & purification*
Polymerase Chain Reaction
Prospective Studies
Sputum / microbiology*
Tuberculosis / epidemiology*
Tuberculosis / microbiology
Young Adult

Substances

DNA, Bacterial

Associated data

ClinicalTrials.gov/NCT02744469

Grants and funding

BdJ and LR were supported by the European Research Council-INTERRUPTB starting grant (nr.311725). SA, DA and CNS were supported by the Third framework agreement programme between DGCD Belgium and ITM, ITM-LRM (FA3-III). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.