Molecular investigations of Mycobacterium tuberculosis genotypes among baseline and follow-up strains circulating in four regions of Eswatini

Abstract

Background: The tuberculosis (TB) epidemic remains a major global health problem and Eswatini is not excluded. Our study investigated the circulating genotypes in Eswatini and compared them at baseline (start of treatment) and follow-up during TB treatment.

Methods: Three hundred and ninety (n = 390) participants were prospectively enrolled from referral clinics and patients who met the inclusion criteria, were included in the study. A total of 103 participants provided specimens at baseline and follow-up within six months. Mycobacterium tuberculosis (M.tb) strains were detected by GeneXpert® MTB/RIF assay (Cephied, USA) and Ziehl -Neelsen (ZN) microscopy respectively at baseline and follow-up time-points respectively. The 206 collected specimens were decontaminated and cultured on BACTEC™ MGIT™ 960 Mycobacteria Culture System (Becton Dickinson, USA). Drug sensitivity testing was performed at both baseline and follow-up time points. Spoligotyping was performed on both baseline and follow-up strains after DNA extraction.

Results: Resistance to at least one first line drug was detected higher at baseline compared to follow-up specimens with most of them developing into multidrug-resistant (MDR)-TB. A total of four lineages and twenty genotypes were detected. The distribution of the lineages varied among the different regions in Eswatini. The Euro-American lineage was the most prevalent with 46.12% (95/206) followed by the East Asian with 24.27% (50/206); Indo-Oceanic at 9.71% (20/206) and Central Asian at 1.94% (4/206). Furthermore, a high proportion of the Beijing genotype at 24.27% (50/206) and S genotype at 16.50% (34/206) were detected. The Beijing genotype was predominant in follow-up specimens collected from the Manzini region with 48.9% (23/47) (p = 0.001). A significant proportion of follow-up specimens developed MDR-TB (p = 0.001) with Beijing being the major genotype in most follow-up specimens (p < 0.000).

Conclusion: Eswatini has a high M.tb genotypic diversity. A significant proportion of the TB infected participants had the Beijing genotype associated with MDR-TB in follow-up specimens and thus indicate community wide transmission.

Keywords: Drug sensitivity testing; Genotypes; Lineages; Multi-drug resistant tuberculosis; Spoligotyping; Tuberculosis.

Fig. 1

Eswatini map (Accessed on 2022 October 17, from: https://geology.com/world/swaziland-satellite-image.shtml)

Fig. 2

Participant flow diagram of baseline and follow-up specimens of patients collected at primary healthcare facilities in Eswatini. A total of n = 103 participants were enrolled into the study and followed through at 6th month

Fig. 3

Spoligotype genotypes cluster analysis at baseline (diagram A) and follow up (diagram B). The figure shows the relationship between lineages and sublineages at baseline (diagram A) and follow-up (diagram B) to note clusters and measure of dissimilarity. Diagram (A) has 2 clusters, where the first cluster has lineage 1,3,1 and orphans and the second cluster is that of only lineage 4. The orphans are closely linked and similar to the lineage 1 EAI1_SOM and EAI5 clades. The second cluster of diagram (A) has only lineage 4 and simplicifolious in H37Rv sub lineage. The two clusters of diagram (A) have very small dissimilarities as the vertical heights are almost equal. The same observation on dissimilarity was made in diagram (B) which also two clusters. In the first cluster, lineage 2 has one clade compared to diagram (A) with two. The orphans are also linked to lineage 1 and 3 clades. The second cluster predominate sub lineage T1 has four clades which are close to X3 sub lineage. Comparing diagram (A) and (B), the measure of dissimilarity is higher in diagram (B)

Fig. 4

Detection of Beijing genotypes in serial isolates. At follow up, most Beijing strains were isolated on the 3^rd, 5^th and 6^th month. The least were detected in the 4^th month