Mycobacterium tuberculosis complex genotypes circulating in Nigeria based on spoligotyping obtained from Ziehl-Neelsen stained slides extracted DNA

Tuberculosis (TB) remains one of the most threatening diseases and Nigeria has one of the world’s largest burdens. This study performed high-throughput spoligotyping directly on sputum smears to describe the Mycobacterium tuberculosis strains circulating among new TB patients in Nigeria. Methods All State TB control programmes in Nigeria were requested to submit 25–50 smear-positive Ziehl-Neelsen (ZN) stained slides for screening during 2013–2014. DNA was extracted from 929 slides for spoligotyping and drug-resistance analysis using microbead-based flow-cytometry suspension arrays. Results Spoligotyping results were obtained for 549 (59.1%) of 929 samples. Lineage 4 Cameroon sublineage (L4.6.2) represented half of the patterns, Mycobacterium africanum (L5 and L6) represented one fifth of the patterns, and all other lineages, including other L4 sublineages, represented one third of the patterns. Sublineage L4.6.2 was mostly identified in the north of the country whereas L5 was mostly observed in the south and L6 was scattered. The spatial distribution of genotypes had genetic geographic gradients. We did not obtain results enabling the detection of drug-resistance mutations. Conclusion/Significance We present the first national snapshot of the M. tuberculosis spoligotypes circulating in Nigeria based on ZN slides. Spoligotyping data can be obtained in a rapid and high-throughput manner with DNA extracted from ZN-stained slides, which may potentially improve our understanding of the genetic epidemiology of TB.
Author summary Using a classical genotyping method designated as “spoligotyping”, which targets polymorphic repetitive DNA loci, we present a general snapshot of the genetic diversity of Mycobacterium tuberculosis complex causing tuberculosis in Nigeria. Our results were obtained on a collection of 549 DNAs, extracted from Ziehl-Neelsen stained sputum smears gathered and representative from 36 Nigerian states and during 2013–2014. We show the ubiquitous presence on the Nigerian territory of a sublineage of Lineage 4, designated as L4.6.2 or “Cameroon” Lineage, which represents almost 50% of all patterns, more prevalent where Mycobacterium africanum west African 1 (Lineage 5) is absent. We also show that Lineage 5 is geographically linked to the south-east of the country, where it is the most prevalent, and represents approximately 20% of all the samples. The last third is linked to all other L4 sublineages and to L6 (M. africanum west African 2). Our results confirm the strong phylogeographical structure of Mycobacterium tuberculosis complex in Nigeria and are suggestive of a long-term coevolution history between Homo sapiens sapiens and Mycobacterium tuberculosis complex.