Publikationsdatenbank

Tuberculosis in animals and humans in Eastern Sudan and the genetic diversity among clinical strains of Mycobacterium tuberculosis complex lineage 3 (2019)

Art

Hochschulschrift

Autor

Shuaib, Yassir Adam (WE 7)

Quelle

Berlin: Mensch und Buch Verlag Berlin, 2019 — v, 127 Seiten

ISBN: 978-3-96729-037-0

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/27665

Kontakt

Institut für Mikrobiologie und Tierseuchen

Robert-von-Ostertag-Str. 7-13
14163 Berlin
+49 30 838 51843 / 66949
mikrobiologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

The Sudan is a high tuberculosis (TB) burden country (i.e. 77 per 100,000 population). Specifically, the eastern part of the country is endemic with high incidence rates of TB reaching up to 275 per 100,000 population during the past decade. Prevalence of Mycobacterium tuberculosis complex (MTBC) strains in ruminants and in humans, as well as potential zoonotic spillovers, are only sparsely investigated. Furthermore, Eastern Sudan is a blind spot with regard to drug resistance rates and virtually no data is available on molecular drug resistance markers of circulating multidrug resistant (MDR) MTBC strains that are resistant to at least isoniazid and rifampicin. Particularly, strains of MTBC lineage 3 (L3), i.e. Delhi/CAS genotype, are highly prevalent in the Sudan and adjacent countries and are the main causative agent of TB in those regions, however, their regional and global genetic diversity and transmission capacity are likely not fully captured yet. Thus, this thesis aims to provide a better understanding of historic and recent transmission dynamics, the genetic background of MTBC L3 strains and to draw a robust phylogenetic framework which will serve future molecular drug resistance assays and whole genome sequencing (WGS) mutation catalogues. First, a total of 2,304 carcasses of ruminants slaughtered at the two slaughterhouses of Kassala were inspected for presence of TB-suggestive lesions (June to November 2014). Second, 383 sputum samples were collected from TB patients at Kassala, Port Sudan, and El-Gadarif teaching hospitals (June to November 2014 and January to July 2016). All collected samples were sent to the National Reference Laboratory (NRL) for Mycobacteria, Borstel in Germany, where all samples were decontaminated and cultured into MGIT liquid medium (BACTEC MGIT 960 system) and onto Löwenstein-Jensen (LJ) and Stonebrink slants. Culture positive samples were subjected to line probe assays and ITS gene sequencing for species identification. Cultures of MTBC strains were subjected to phenotypic drug susceptibility testing (pDST), and MTBC DNA was used for genotypic characterisation (genotyping) using spoligotyping, 24-loci MIRU-VNTR typing, and WGS. Culture-negative and contaminated samples were subjected to an in-house real-time PCR (qPCR) for detection of mycobacterial DNA. Finally, to get insights into MTBC L3 global population structure, a data set containing the results of 24-loci MIRU-VNTR genotyping of 1,685 strains from 22 countries across Asia, Africa, and Europe was analyzed. The pDST of 1,070 of the strains was also analyzed. A total of 159 out of the 1,685 L3 strains representing a wide global diversity were selected for a WGS analysis. This work showed that only 0.1% (2/2304) of the carcasses of ruminants were found with TB-suggestive lesions. None of the MTBC members was isolated from the collected TB-suggestive lesions but a slow growing Mycobacterium species with the closest similarity to M. terrae group. Furthermore, growth of organisms was obtained from 51.2% (196/383) of TB patients derived sputum samples from Eastern Sudan to identify the MTBC lineage of the infecting strains, molecular clusters as surrogate marker for recent transmission in the region, and drug resistance profiles. Of the isolated bacteria, 87.2% (171/196) were identified as M. tuberculosis and 7.2% (14/196) as M. intracellulare. The remaining 5.6% (11/196) positive cultures had more than one species of bacteria. The qPCR showed that 75.1% (127/169) of the culture-negative and contaminated samples were positive for MTBC DNA and 4.7% (8/169) for nontuberculous mycobacteria (NTM) DNA. Results of culture and qPCR together showed a positive predictive value of 90.7% (331/365) for smear microscopy for detection of mycobacteria in Eastern Sudan. Moreover, MTBC molecular analysis revealed that 73.4% (130/177) belong to L3, 22.5% (40/177) to L4, 2.8% (5/177) to L1, and 1.1% (2/177) to L2. pDST data were available for 96.7% (175/181) of the MTBC strains. A percentage of 22.3% (39/175) of the strains with pDST results available showed resistance to at least one of the tested first line antibiotics, of which 10.3% (18/175) were MDR strains. Furthermore, 77.8% (n=14) of the MDR strains were clustered. Overall MTBC L3 strains are divided into six Delhi/CAS Miru Groups (DCMGs) and into eight WGS-based groups. Each of the eight groups has specific mutations. Of the L3 strains with available pDST data, 21.5% were MDR. This thesis suggests that the overall prevalence of MTBC strains in TB-suggestive lesions is very low in carcasses of ruminants in Kassala and zoonotic transmission of MTBC bacteria is unlikely. It further suggests the need for more specific laboratory tests such as the Xpert MTB/RIF to avoid possible overtreatment as some sputum samples did not contain MTBC bacteria and few others had only NTMs. Moreover, this work confirmed that L3 strains are responsible for causing the majority of TB cases in Eastern Sudan as well as revealed transmission of MDR MTBC strains. Poverty and weaknesses in TB control and diagnostics are potential factors that lead to the emergence of MDR MTBC transmission in the country. This needs to be carefully traced in the following years and implementation of rapid molecular DSTs should be considered. Indeed, the findings of this work provided a robust phylogenetic classification scheme for L3 strains for future studies by defining six distinct DCMGs and eight WGS-based groups. The defined DCMGs have also distinct spatial distribution. It is most likely that L3 strains emerged in South Asia (with L3.2 as the most ancestral strains) and subsequently spread successfully into other parts of the world. Historic migration waves but also recent transmission likely shaped the MTBC L3 phylogeny. However, the effect of antibiotic treatment is moderate, compared to MTBC L2 strains for instance, and it affects all defined DCMGs equally, except DCMG5 which showed a lower MDR MTBC proportion. Nevertheless, more than half of the MDR MTBC L3 strains were related to recent transmission rendering MTBC L3 strains the possible driver of MDR TB in East Africa. Jointly, this work showed that zoonotic spillovers of MTBC strains virtually don’t occur, and MTBC L3 strains are the main causative agent of TB and are responsible of all MDR TB cases in Eastern Sudan. Moreover, the global genetic analysis of MTBC L3 strains from 22 different countries revealed that the MTBC L3’s genetic diversity is higher than currently expected. This finding has significantly improved the classification system and will serve future studies on molecular drug resistance determinants. The finding further provided insights in the evolutionary history of this regionally dominating MTBC lineage.