Recent advances in molecular strain typing such as the development of 24-loci MIRU-VNTR typing provide a powerful tool to analyze MTBC population structure and transmission dynamics locally and on the global level, which provides valuable information for the development of effective tuberculosis control policy. In this study, we present the first in-depth analysis of the population structure of M. tuberculosis strains in Northwest Ethiopia based on high-resolution MIRU-VNTR 24-loci typing and spoligotyping. Our data confirm a highly diverse population structure that comprises, thirteen phylogenetic lineages, four of which were not described before. Furthermore, our data indicate a high rate of recent transmission, of which the spread of resistant and MDR strains is of special importance.
While homoplasy is a true phenomenon within the evolution of TB, spoligotyping has been shown to provide invalid phylogenetic classifications by suggesting homoplasy too often . In the contrary, the MIRU-VNTR 24-loci typing method applied in our study has the advantage to allow for high-resolution genotyping needed for molecular epidemiological studies and, simultaneously, for valid phylogenetic strain classification enabling screening for new phylogenetic lineages/clonal complexes .
Using this method, 90.6% of the strains investigated were classified into various M. tuberculosis complex lineages; of which, 58.9% were described before and 31.6% were newly described in this study. We documented that M. tuberculosis Dehli/CAS is the predominant phylogenetic lineage in Ethiopia, accounting for 39% of investigated strains. Similarly, a previously published study from the capital city of Ethiopia showed that 43.5% of the strains were of the CAS lineage , and a study from Sudan  also showed that M. tuberculosis Dehli/CAS is the predominant lineage (49%) of investigated strains. The Dehli/CAS lineage is essentially localized in the Central Asia and Middle-East, more specifically in India . Two hypotheses could explain the presence of high Dehli/CAS lineage in Ethiopia: (i) the large Indian and Chinese communities in Ethiopia due to the growing economic partnerships between Ethiopia and the two Asian countries, India and China may have contributed in the introduction of this lineage; or (ii) this lineage could have emerged from Ethiopia and migrated through Asia, this hypothesis is in agreement with the suggestion that East Africa is the origin of M. tuberculosis complex species .
Additionally, we confirmed the presence of previously undefined phylogenetic lineages named as Ethiopia_3, Ethiopia_1, Ethiopia_H37RV-like and Ethiopia_2 that were clearly defined by tree based, as well as by minimum spanning tree-based analysis. However, comparison with other studies is hampered by the fact that they are mainly based on IS6110 DNA fingerprint and/or spoligotyping analysis hindering a valid analysis of the population structure and standardized comparisons based on MIRU-VNTR nomenclature. Thus, the actual picture of M. tuberculosis population diversity in African, high-incidence settings is largely incomplete and needs a systematic investigation with phylogenetic useful genotyping methods.
This study also showed a significant association between infection with strains of the Haarlem lineage and multi-drug resistance, resistance to all first line anti-TB drugs and resistance to each first line anti-TB drugs including INH, RMP, STM, EMB and PZA. Similarly, a previous study from Tunisia showed that the Haarlem family genotype has a similar relationship with drug resistance and rapid clonal expansion . From TB-control point of view, it is relevant to understand whether specific genotype families are overrepresented among drug-resistant cases and, in particular, if these resistant strains are successfully transmitted within the community. In this study, HIV infection was not significantly associated with resistance to anti-TB drugs. The high HIV prevalence in the study subjects did not appear to be a significant risk factor selectively driving drug resistance development and transmission. This might be due to the fact that HIV infection increases the susceptibility of the population for both drug susceptible and drug resistant M. tuberculosis strains.
Clustering is a marker for recent transmission [30–32]. By using degree of recent TB transmission in a study population, one can estimate the efficacy of the TB control program . Both high TB incidence and the current drug-resistance rates in Ethiopia are indicative of defects of the TB control program [2, 4, 16]. Supporting this suggestion, we found a high rate of clustering, 45.1% of the total strains investigated. This is in agreement with the previous reports from the capital city of Ethiopia that showed clustering rate of 41.2%  and 48.1% .
Even more important, we confirm an elevated clustering rate in drug resistant strains in general as well as for MDR strains. Similarly, there was a significant association between recent transmission and patients with the history of previous TB treatment, infection with INH resistant strains, STM resistant strains, EMB resistant strains, strains resistant to one or more first line anti-TB drugs and patients with strains resistant to all first line anti-TB drugs. This might be due to the fact that, in Ethiopia there is no culture and drug susceptibility testing facility for routine diagnosis of drug resistance, thus, drug resistant-TB is only diagnosed after prolonged treatment with first-line anti-TB drugs and clinical recognition that treatment has failed. Treatment of drug-resistant TB with standard first line drugs, instead of a regimen designed according to the resistance pattern has several potential adverse consequences: patients remain on inadequate treatment longer, increasing the risk of treatment failure or death; selection of drug resistant strains and patients remain infectious, promoting transmission to close contacts . These data indicate a successful transmission of drug resistant and MDR strains in the community, a situation that needs to be carefully monitored in the future to determine extensive transmission of resistant strains early enough to avoid more significant problems for TB control as already eminent in several parts of Eastern Europe or South Africa [34, 35].
Interestingly, we present evidence of significant association between recent transmission and the Dehli/CAS, Ethiopia_3, TUR and Ethiopia_H37Rv like strain infections. Similarly, Gagneux et al. have recently proposed that the major M. tuberculosis lineages have evolved so as to become adapted to specific host genetic backgrounds and are much more likely to transmit and cause disease among patients of the same ethnicity .