First insights on the genetic diversity of MDR Mycobacterium tuberculosis in Lebanon

Tuberculosis (TB) has been a global health problem for millennia and still causes epidemics worldwide. According to the 2017 WHO Global TB Report, at least 10.4 million people fell ill with TB in 2016 [1]. For the past five years, it ranked as the leading cause of death from a single infectious agent, causing more mortalities than HIV/AIDS [1]. A major worldwide threat is the emergence of drug-resistant tuberculosis (DR-TB) and multidrug-resistant TB (MDR-TB) carrying resistance to both isoniazid and rifampicin and even extensively drug resistant TB (XDR-TB) [2]. TB comprises seven lineages [3]. Lineages 1 and 3 are limited to East Africa and parts of Asia. Lineage 2, known as the East-Asian lineage, includes the Beijing family of strains and predominates in East Asia. Lineage 4, known as the Euro-American lineage, is present in Asia, Europe, Africa and America while lineages 5–7 are geographically restricted to specific regions in Africa [4].

In Lebanon, incidences of TB are on the rise since 2006 with increasing mortality rates, especially among HIV-positive patients [1, 5]. A total of 860 people in Lebanon were infected with TB in 2016 [1]. With a lack of studies in Lebanon on TB epidemiology, burden and genetic diversity, there is a need to address TB influx patterns. Lebanon hosts, in addition to migrants from Syria, workers coming from Bangladesh, Pakistan, Philippines, Russian Federation, Vietnam, Ethiopia, India and other origins, which are among the highest 30 countries with MDR-TB incidences worldwide [1]. The genetic lineages of TB circulating in Lebanon were not addressed previously. Despite increasing incidences of TB in the country [1, 5], the “End TB Strategy” milestones set by the WHO are to reduce the mortality rates caused by TB by 35% in 2020, 75% in 2025 and by 95% in 2035 compared to the 2015 baseline values [1]. Thus, there is a definite need for more rapid and comprehensive diagnostics tools to improve treatment outcomes and epidemiological tracing to limit TB spread [6].

With decreasing costs, increasing availability and ease of usage [7], whole genome sequencing (WGS) provides unprecedented opportunities to obtain reliable predictions of drug susceptibilities and accurate identification of genetic polymorphisms such as single nucleotide polymorphisms (SNPs) and small insertions and deletions (indels) affecting drug resistance in TB [8, 9]. Analysis of genetic markers provided from WGS was previously used to distinguish persistent infections from MDR-TB and XDR-TB reinfections [10, 11]. Moreover, integrating WGS with patients’ information was successful in identifying transmission events and super-spreaders of infections [12‐14]. It was also used to overcome the limited resolution obtained from the mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) molecular characterization method [15], and to highlight several outbreak scenarios that were previously missed by standard routine techniques [7]. End-to-end WGS-based diagnostic schemes and semi-automated pipelines for TB analysis have already shown promise in replacing traditional TB diagnostic tools in Europe and North America [16‐18]. Another advantage of WGS is the direct sequencing of TB from sputum samples using culture-free approaches [19].

With the absence of genomic studies characterizing TB strains circulating in the Middle East, in this warranted pilot study, we performed WGS on 13 MDR-TB and susceptible-TB isolates collected from Lebanon to gain insights on the distribution of genetic lineages of TB, examine their genetic basis of drug resistance, and decipher mutations involved in antibiotic resistance genes. In order to gain a deeper understanding of the genomic diversity and evolution of TB in Lebanon, genomic profiling and phylogenetic analysis at the single nucleotide variants (SNV) level were conducted to accurately evaluate the position of local TB cases with respect to global reference genomes. The data obtained provides the first perceptions on the genetic composition of TB lineages circulating in the Middle East and confirms on the utility of WGS as a valuable technique in routine clinical diagnosis.

This is the first report on the diversity of M. tuberculosis lineages circulating in Lebanon and in the region. In our collection of isolates, we identified types covering the most common global lineages, which revealed the heterogeneity within TB causing isolates in Lebanon. Our findings reveal that the Euro/American lineage (Lineage 4) was the most dominant in Lebanon (53.8%), followed by the M. bovis (15.4%) and Delhi/Central Asia (Lineage 1) (15.4%), the Beijing/East Asia (Lineage 2) (7.7%), and the East Africa/Indian Ocean lineage (Lineage 3) (7.7%). SNV-based phylogenetic analysis grouped the isolates in agreement with their assigned lineages with a higher accuracy than IS6110 mapping and spoligotyping. The European/American lineage was found to be prevalent in several North African countries including Morocco, Tunisia, Algeria and Libya [35].

The MDR isolates belonged to the Beijing/East Asia lineage (Lineage 2) (TB-17), while the two other MDRs belonged to the Delhi/Central Asia lineage (Lineage 1) (TB-12 and TB-16). In fact, TB belonging to the Beijing/East Asian lineage was found to be driving the massive spread of MDR TB in Eurasia [36, 37]. They were associated with large MDR TB outbreaks [38] and appear to be rapidly expanding in population size [39] due to their hypermutability [40], increased transmissibility, and hypervirulence compared to other MTBC lineage [41]. On the other hand, the Delhi/Central Asia lineage is the second predominant genotype in pre-XDR and XDR TB following the Beijing/East Asian lineage [42]. The Delhi/Central Asia lineage was detected in the Middle East and Central Asia and preferentially in India [43].

In this study, 15.4% (n = 2) of the isolates belonged to the M. bovis lineage (TB-5 and TB-8) and had different spoligotypes. They showed one IS6110 compared to an average of 9.5 IS6110 in the other isolates. IS6110 copy number ranges from 0 to 27, with a mean of approximately 13 in the seven TB lineages [34]. Differing levels of IS could be related to TB host adaptability [44]. Yet, to date, IS6110 copy number was not explored in M. bovis although it was considerably less than that in M. tuberculosis, with typically just one insertion noted [45], as also observed in our study. Interestingly, a unique single transposable IS6110 upstream of the phoPR operon increased its expression, thereby facilitating the dissemination of M. bovis in the human host [46]. Scarce data on M. bovis exist in Lebanon with the prevalence being 3.3% [47]. The consumption of raw milk appears to be a common source of transmission [48], and the burden of M. bovis is underestimated in human beings [49]. Symptomatic and asymptomatic animals serve as reservoirs of M. bovis and favor its dissemination to humans and wildlife species [50]. The accurate diagnosis and treatment of M. bovis in humans is necessary to achieve a world free of TB by 2035 as part of the WHO’s end TB strategy [51].

Multidrug-Resistant TB (MDR-TB) isolates belonging to the Delhi (n = 2) lineage and the Beijing (n = 1) lineage clustered significantly far from one another based on their phylogenetic SNV grouping. Two SNPs, noted as SNP761155CT and SNP2155168CG were detected in all three of the MDR isolates. These characteristic point mutations are causative of changes in rpoB S450 L and katG S315 T respectively. De novo generation of drug resistant TB is a major healthcare concern, as the incidence, treatment days, treatment expenses, and mortality rates would all increase with the spontaneous generation of resistant strains immune to the very few available effective antimicrobial agents.

By comparing the spoligotype and VNTR fingerprints of the isolates with the SNV phylogenetic clustering, some of the isolates in this study showed nearly identical VNTR patterns and spoligotype fingerprints yet appeared to be phylogenetically unrelated. Also, the SNV-based phylogenetic analysis was also more accurate in grouping isolates to lineages compared to IS6110 that in the case of our isolates did not accurately assign two isolates belonging to lineage 4. This revealed the presence of genome-based differences at regions not linked to those targeted by the aforementioned classical typing methods. Other advantages of WGS include outbreak analysis and surveillance [52] and the sequencing from uncultured sputa of patients with diagnosis-ready data generated in just a few days from sample collection [19].

Although the small number of studied samples provided some insights into the heterogeneity of TB circulating in Lebanon, it may not represent all the genetic diversity of TB in the region and the collection of a larger sample size is necessary. Thus, our efforts will include a broader sampling that includes Lebanon and neighboring countries. As a further step forward, we push for the analysis of the intra-host evolution of MDR-TB isolates to prevent treatment failure and decrease mortality rates. Reinfection by MDR-TB or relapse is of critical importance, thus the detection of de novo generated SNVs especially in HIV positive patients is of paramount importance [53].

As highlighted very recently, the burden of TB has been a major public health issue in Lebanon. The Syrian humanitarian crisis completes its 8th year in succession, continuously adding to the weight of the problem at hand [54]. With over 600 new cases arising per year in the last decade, and the scarcity of studies that shed light on the evolution of TB in the region [5], Lebanon has been held back from joining the global efforts to fight TB [1]. Taking into account the small size of Lebanon, the genomic heterogeneity of mycobacteria can only be understood and better dealt with by high quality differentiation and high throughput methods.

If we are to decrease incidences to the WHO specified pre-elimination limit of < 10 TB cases per million by 2035, and < 1 TB case per million by 2050, TB incidences have to drop by 90% in the following decade, and by 99% in the year 2050 [1]. Thus, we recommend more stringent TB testing of foreign workers prior to their travel to Lebanon, and push towards the implementation of WGS as a core diagnostic tool for TB management over the coming years.