Virulence of novel East African mycobacterium tuberculosis lineages

Tuberculosis (TB) poses a major public health threat in low welfare countries. Most cases and deaths from TB occur in Asia and sub Saharan Africa. A deeply rooted phylogenetic lineage of Mycobacterium tuberculosis (Mtb) termed lineage 7 was recently discovered in the Amhara Region of Ethiopia, bordering Sudan and South Sudan. In the EvoTB project, Ethiopia, Sudan and South Sudan are currently collaborating to characterize the novel East African Mtb genotype and its virulence traits. We have deciphered the clinical and transmission relevance of Mtb lineage 7 strains in the Horn of Africa. Objective 1) We have characterized the predominant strains of Mtb circulating in Ethiopia by VNTR typing and whole genome sequencing. We investigated the genetic diversity and association of specific Mtb lineages with sociodemographic and clinical parameters among pulmonary TB patients in the Amhara Region of Ethiopia. The study revealed a high diversity of Mtb lineages, among which approximately 25% were not previously reported. The largest cluster belonged to the lineage Central Asian (CAS) (26.0%), the second largest to lineage 7 (15.6%), and the third largest to the lineage Haarlem (15.2%). Also, four sub-lineages were new in the MIRU-VNTRplus database. Objective 2) We have investigated the association of the Mtb lineages with transmissibility of infection, clinical outcome and severity of disease. Notably, a patient delay in seeking treatment was significantly longer among patients infected with lineage 7 strains than in patients infected with CAS strains (adjusted odds ratio, 4.7; 95% confidence interval, 1.6 to 13.5). The result also showed that lineage 7 strains grew more slowly than other Mtb strains. Cases of Haarlem and NW-ETH3 infections appeared in defined clusters and were more likely to demonstrate resistance to streptomycin and any of the first-line anti-TB drugs compared to other lineages. Objective 3) We have investigated the association of the Mtb lineages with drug resistance to first line anti-TB drugs, identifying sensitive, mono-resistant and multi-drug resistant Mtb strains. Only 2% of these Mtb strains were MDR. Objective 4) We have performed whole genome sequencing (WGS) of selected strains of the Mtb lineage 7 as well as all the other lineages. We performed WGS of 30 lineage 7 strains from Ethiopian patients. More than 800 mutations specific to Mtb lineage 7 strains were identified. A total of 22346 bp deletions were observed, and the proportion of non-synonymous single nucleotide polymorphisms in 3R genes was considerably higher after the recent expansion of Mtb lineage 7 strains started. This event coincided with In addition, mass spectrometry based proteomic analysis was performed to compare the protein profiles of Mtb lineage 7 strains and the Mtb lineage 4 reference strain H37Rv. A total of 2867 protein covering 71% of the predicted Mtb proteome were identified. A number of Mtb interactive proteins groups involved in growth and virulence were less abundant in lineage 7 compared to lineage 4. Six components of the type 7 secretion system ESX-3 proteins involved in iron acquisition were less abundant in Mtb lineage 7 compared to lineage 4. The EvoTB project has provided important contributions to TB control in Ethiopia. Notably, the patients infected with Mtb of lineage 7 had a significantly late health seeking behavior. This diagnostic delay has implications for silent TB cases, increasing the risk of transmission to uninfected individuals. Furthermore, the considerable clustering of Mtb cases indicates ongoing transmission in the study area. These findings inform policy makers involved in TB control program to intensify efforts to identify and treat active cases. Our findings thus provide novel biomarkers and inform TB control policy makers on the importance of expanding the capacity of TB culture and drug susceptibility testing facilities to combat the spread of drug-resistant TB in the region. The EvoTB project has provided important contributions to TB control and innovation based on the Mtb lineage 7 strains from Ethiopia. Notably, the patients infected with Mtb of lineage 7 had a significantly late health seeking behavior. This diagnostic delay has implications to silent TB cases, increasing the risk of transmission to uninfected individuals. Haarlem strains frequently demonstrated resistance to one or more first-line anti-TB drugs. Furthermore, the considerable clustering of Mtb cases indicates ongoing transmission in the study area. These findings inform policy makers involved in TB control program to intensify efforts to identify and treat active cases. Intensified active case finding and contact tracing activities are crucial to expedite diagnosis and treatment of TB, which ultimately results in reduction of TB transmission. The study area, Amhara Region, has more than 20 million inhabitants, and there is only one TB culture facility in the region. Our findings thus inform TB control policy makers on the importance of expanding the capabilities for TB culture and drug susceptibility testing facilities to combat the spread of drug-resistant TB in the region. The EvoTB study also identified unique genomic signatures associated with Mtb lineage 7 strains and identified SNPs in genes related to both the clinical and microbiological features. The relatively high proportion of nsSNPs in 3R genes may have contributed to and explain the phylogenetic expansion of lineage 7 strains that started approximately 310 years ago. The high proportion of nsSNPs and deletions observed in specific genes may have contributed to phenotypic consequences including slow growth. These findings will be used as a basis to conduct functional biochemical studies and delineate the relative association to virulence and the relation to clinical presentation as well as biomarkers in diagnostics. As a highly contemporary clinical microbiology lab we applied electrospray-based mass spectrometry (MS) analysis to characterize the Mtb lineage 7 strains in detail. The MS analysis provided unique insight into the lineage 7-specific protein profile which provides cues to understanding the differential properties of lineage 7 strains in terms of slow growth, fitness for survival and pathogenesis. Specifically, the down-regulation of the type 7 secretion system ESX-3 proteins involved in iron acquisition will be used as a basis for further study of lineage 7 as well as for development of highly warranted novel sub-species-specific diagnostic tests. Increased understanding of how Mtb acquires and uses iron during infection could lead to the development of new antibiotics to be used in treating TB, which would be of great benefit for patients, particularly in low welfare countries. In addition to developing novel diagnostics, we are advancing towards antibiotic resistance testing, direct specimen exploration and typing as well as using the reduced growth characterized as a potential new drug target. Overall, the application of MS in our research has enabled our group to become among the leading scientists in the field of clinical mycobacteriology. The new EvoTB knowledge has further created and strengthened networks between clinical microbiologists, basic scientists and allied professions and built multiple capacity-facilitating applications and diagnostic opportunities in this multidisciplinary field across Ethiopia and Norway.