Tuberculosis Complex Strains Research Articles

Transmission patterns of rifampicin resistant Mycobacterium tuberculosis complex strains in Cameroon: a genomic epidemiological study

BackgroundDetermining factors affecting the transmission of rifampicin (RR) and multidrug-resistant (MDR) Mycobacterium tuberculosis complex strains under standardized tuberculosis (TB) treatment is key to control TB and prevent the evolution of drug resistance.MethodsWe combined bacterial whole genome sequencing (WGS) and epidemiological investigations for 37% (n = 195) of all RR/MDR-TB patients in Cameroon (2012–2015) to identify factors associated with recent transmission.ResultsPatients infected with a strain resistant to high-dose isoniazid, and ethambutol had 7.4 (95% CI 2.6–21.4), and 2.4 (95% CI 1.2–4.8) times increased odds of being in a WGS-cluster, a surrogate for recent transmission. Furthermore, age between 30 and 50 was positively correlated with recent transmission (adjusted OR 3.8, 95% CI 1.3–11.4). We found high drug-resistance proportions against three drugs used in the short standardized MDR-TB regimen in Cameroon, i.e. high-dose isoniazid (77.4%), ethambutol (56.9%), and pyrazinamide (43.1%). Virtually all strains were susceptible to fluoroquinolones, kanamycin, and clofazimine, and treatment outcomes were mostly favourable (87.5%).ConclusionPre-existing resistance to high-dose isoniazid, and ethambutol is associated with recent transmission of RR/MDR strains in our study. A possible contributing factor for this observation is the absence of universal drug susceptibility testing in Cameroon, likely resulting in prolonged exposure of new RR/MDR-TB patients to sub-optimal or failing first-line drug regimens.

Multi-Resistant Tuberculosis in Western Algeria: about 36 Cases

Multi-drug resistant tuberculosis (MDR-TB) is defined by the loss of susceptibility of Mycobacterium tuberculosis complex strains to the two major anti-tuberculosis drugs: isoniazid (H) and Rifampicin (R). This resistance may be acquired or primary. The aim of our study was to describe the epidemiological, clinical, bacteriological and evolutionary profile of multi-resistant tuberculosis patients in Western Algeria. To do so, aretrospective analytical study, which included 36 patients, was carried out in the pneumo-phthisiology department (B) of the Oran University Hospital (West Algeria) from January 2010 to December 2014. Our result showed an average age of 33.7 years. The risk factors for MDR-TB highlighted were tobacco (n=12; 33.3%), alcoholism (n=1; 2.8%) and family history of MDR-TB (n=3; 8.33). Primary MDR-TB was present in 11.11% of cases and secondary MDR-TB in 88.89% of cases. There was resistance to H and R in 13.8% of cases (n=5); to H, R and Streptomycin (S) in 50% of cases (n=18); to H, R and Ethambutol (E) in 5.6% of cases (n=2). The percentage of patients who had lost the sensitivity of the Bacillus to the four first-line anti-tuberculosis drugs (H, R, S, and E) was 25% of all patients tested (n=9) and H, R, S, and Ofloxacin (O) in one case. Five patients died. The rate of primary resistance found in this study constitutes a threat to efforts to control MDR tuberculosis in Algeria. It is therefore important to update the results and assess the extent of the problem.

ESX-1-Independent Horizontal Gene Transfer by Mycobacterium tuberculosis Complex Strains.

ABSTRACTCurrent models of horizontal gene transfer (HGT) in mycobacteria are based on “distributive conjugal transfer” (DCT), an HGT type described in the fast-growing, saprophytic model organism Mycobacterium smegmatis, which creates genome mosaicism in resulting strains and depends on an ESX-1 type VII secretion system. In contrast, only few data on interstrain DNA transfer are available for tuberculosis-causing mycobacteria, for which chromosomal DNA transfer between two Mycobacterium canettii strains was reported, a process which, however, was not observed for Mycobacterium tuberculosis strains. Here, we have studied a wide range of human- and animal-adapted members of the Mycobacterium tuberculosis complex (MTBC) using an optimized filter-based mating assay together with three selected strains of M. canettii that acted as DNA recipients. Unlike in previous approaches, we obtained a high yield of thousands of recombinants containing transferred chromosomal DNA fragments from various MTBC donor strains, as confirmed by whole-genome sequence analysis of 38 randomly selected clones. While the genome organizations of the obtained recombinants showed mosaicisms of donor DNA fragments randomly integrated into a recipient genome backbone, reminiscent of those described as being the result of ESX-1-mediated DCT in M. smegmatis, we observed similar transfer efficiencies when ESX-1-deficient donor and/or recipient mutants were used, arguing that in tubercle bacilli, HGT is an ESX-1-independent process. These findings provide new insights into the genetic events driving the pathoevolution of M. tuberculosis and radically change our perception of HGT in mycobacteria, particularly for those species that show recombinogenic population structures despite the natural absence of ESX-1 secretion systems.

Microevolution of Mycobacterium tuberculosis Subpopulations and Heteroresistance in a Patient Receiving 27 Years of Tuberculosis Treatment in Germany.

ABSTRACTPreexisting and newly emerging resistant pathogen subpopulations (heteroresistance) are potential risk factors for treatment failure of multi/extensively drug resistant (MDR/XDR) tuberculosis (TB). Intrapatient evolutionary dynamics of Mycobacterium tuberculosis complex (Mtbc) strains and their implications on treatment outcomes are still not completely understood. To elucidate how Mtbc strains escape therapy, we analyzed 13 serial isolates from a German patient by whole-genome sequencing. Sequencing data were compared with phenotypic drug susceptibility profiles and the patient’s collective 27-year treatment history to further elucidate factors fostering intrapatient resistance evolution. The patient endured five distinct TB episodes, ending in resistance to 16 drugs and a nearly untreatable XDR-TB infection. The first isolate obtained, during the patient’s 5th TB episode, presented fixed resistance mutations to 7 anti-TB drugs, including isoniazid, rifampin, streptomycin, pyrazinamide, prothionamide, para-aminosalicylic acid, and cycloserine-terizidone. Over the next 13 years, a dynamic evolution with coexisting, heterogeneous subpopulations was observed in 6 out of 13 sequential bacterial isolates. The emergence of drug-resistant subpopulations coincided with frequent changes in treatment regimens, which often included two or fewer active compounds. This evolutionary arms race between competing subpopulations ultimately resulted in the fixation of a single XDR variant. Our data demonstrate the complex intrapatient microevolution of Mtbc subpopulations during failing MDR/XDR-TB treatment. Designing effective treatment regimens based on rapid detection of (hetero) resistance is key to avoid resistance development and treatment failure.

Burden of Drug resistant Tuberculosis in newly diagnosed Tuber-culosis patients of Khyber Pakhtunkhwa, Pakistan

Abstract Objective: To explore the burden of drug resistant tuberculosis in Khyber Pakhtunkhwa, Pakistan. Methods: A total of 8220 referred suspected TB patient samples were collected from 1st March 2016 to 28th February 2017 from TB laboratories across all districts of Khyber Pakhtunkhwa province and processed for the detection of drug resistant tuberculosis (DR-TB). Drug Susceptibility testing (DST) on all Mycobacterium tuberculosis complex (MTC) strains was performed at Provincial TB Reference Laboratory, Peshawar. Results: Among the 8220 suspected TB samples, 1351 samples were declared positive on culture. The DST results showed that 525 samples were resistant to at least one of the first and second line anti-TB drugs. Importantly, among these 525 TB resistance cases, XDR-TB were 0.4% (n=5), MDR-TB were 4.6% (n=62), polyresistant were 18% (n=243), monoresistant was 16% (n=215) and 61% (n=826) were reported as pan-sensitive. Gender wise ratio are slightly increased in female (n= 4230; 51.5%) as compared to male (n=3990; 48.5%), while the age group 15-24 were the main target of TB disease (n=1978; 24%). Conclusion: Our data suggests that DR-TB cases especially MDR-TB and XDR-TB in newly diagnosed TB patients are alarmingly increased in Khyber Pakhtunkhwa region. Furthermore, the timely detection of drug resistance is important to optimize treatment and management of MDR-TB as well as XDR-TB. Key words: Tuberculosis; MDR-TB; XDR-TB; Continuous....

Transmission of pre-XDR and XDR-TB in the Mumbai Metropolitan Region, India

Background: Multidrug-resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis complex (MTBC) strains are a great challenge for tuberculosis (TB) control in India. Still, factors driving the MDR/XDR epidemic in India are not well defined. Methods: Whole genome sequencing (WGS) data from 1 852 MTBC strains obtained from patients from a tertiary care hospital laboratory in Mumbai were used for phylogenetic strain classification, resistance prediction, and cluster analysis (12 allele distance threshold). Factors associated with pre-XDR/XDR-TB were defined by odds ratios and a multivariate logistic regression model. Results: 1 017 MTBC strains were MDR, out of which 57.8 % (n=591) were pre-XDR, and 17.9 % (n=183) were XDR. Lineage 2 (L2) strains represented 41.7 % of the MDR, 77.2 % of the pre-XDR, and 86.3 % of the XDR strains, and were significantly associated with pre-XDR/XDR-TB (P < 0.001). Cluster rates were high among MDR (78 %) and pre-XDR/XDR (85 %) strains with three dominant L2 strain clusters (Cl 1-3) representing half of the pre-XDR and two thirds of the XDR-TB cases. Cl 1 strains accounted for 52.5 % of the XDR MTBC strains. Transmission could be confirmed by identical mutation patterns of particular pre-XDR/XDR strains. Conclusions: High rates of pre-XDR/XDR strains among MDR-TB patients require rapid changes in treatment and control strategies. Transmission of particular pre-XDR/XDR L2 strains is the main driver of the pre-XDR/XDR-TB epidemic. Accordingly, control of the epidemic in the region requires measures with stopping transmission esp. of pre-XDR/XDR L2 strains. Funding Statement: Parts of this work have been supported within the CRYPTIC consortium by the Bill & Melinda Gates Foundation [OPP1133541] and the Wellcome Trust [200205/Z/15/Z], the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germanys Excellence Strategy – EXC 2167 Precision Medicine in Inflammation, and the Leibniz Science Campus Evolutionary Medicine of the LUNG (EvoLUNG). Declaration of Interests: None to declare. Ethics Approval Statement: Approval for the CRyPTIC study was obtained from the Health Ministry’s Screening Committee (HMSC), Government of India dated 6th October 2016. Approval was also obtained from the Institutional Ethics Committee (IEC) of The Foundation for Medical Research, Mumbai (Ref nos. FMR/IEC/TB/01a/2015 and FMR/IEC/TB/01b/2015) and Institutional Review Board of P.D. Hinduja Hospital and Medical Research Centre, Mumbai (Ref no. 915-15-CR [MRC]).

Türkiyenin Doğusunda Çeşitli Klinik Örneklerden İzole Edilen Mycobacterium tuberculosis Suşlarinin Major Anti-Tüberküloz İlaçlara Duyarlılıklarının Araştırılması

Objective: In the study, 419 Mycobacterium tuberculosis complex strains (MTC), which were isolated from samples sent to the Regional Tuberculosis Laboratory in our city between 2015 and 2019 with suspected tuberculosis from Erzurum and surrounding cities were investigated. Methods: Our research is a retrospective cross-sectional study and clinical samples sent to the Regional Public Health Laboratory with suspicion of Tuberculosis within a five-year period from January 2015 to December 2019 were first evaluated by using Ehrlich-Ziehl-Neelsen staining technique. The samples were then simultaneously inoculated into the automated culture system (BACTEC MGIT 960) with Löwenstein-Jensen medium. Sensitivity research was conducted for isoniazide, rifampicin, streptomycin and ethambutol antibiotics with BACTEC MGIT 960 system to samples defined by MTC identification test (TBc ID) from reproduction positive tubes. Results: In our study, 337 (80.4%) of the 419 MTC strains investigated in our study were susceptible to all major anti-TB drugs, while 82 (19.6%) were found to be resistant to at least one major anti-TB drug. The distribution of resistance ratios was isoniazid (11.9%), streptomycin (11.7%), rifampicin (4.1%), ethambutol (3.6%). Multi-drug resistant (MDR)–TB ratio (isoniazid+ rifampicin) was found to be (3.6%). Conclusions: In our study, in the strains in which resistance to anti-TB drugs was investigated, the highest drug resistance was determined for isoniazid. The lowest drug resistance was determined for ethambutol.

Broad diversity of Mycobacterium tuberculosis complex strains isolated from humans and cattle in Northern Algeria suggests a zoonotic transmission cycle.

Mycobacterium tuberculosis complex (MTBC) comprises closely related species responsible for human and animal tuberculosis (TB). Efficient species determination is useful for epidemiological purposes, especially for the elucidation of the zoonotic contribution. In Algeria, data on MTBC genotypes are largely unknown. In this study, we aimed to investigate the occurrence and diversity of MTBC genotypes causing human and bovine TB in Northern Algeria. During a two-year sampling period (2017–2019) in two regions of Northern Algeria, we observed an overall prevalence of 6.5% of tuberculosis (TB) among slaughtered cattle, which is higher than previous Algerian data yet comparable to neighboring countries. A total of 296 Mycobacterium tuberculosis complex (MTBC) isolates were genotyped by spoligotyping: 181 from tissues with TB-like lesions collected from 181 cattle carcasses and 115 from TB patients. In human isolates, we identified 107 M. tuberculosis, seven M. bovis and one “M. pinnipedii-like”, while for bovine samples, 174 isolates were identified as M. bovis, three as M. caprae, three as “M. pinnipedii-like” and one as “M. microti-like”. The majority of isolates (89.2%) belonged to 72 different known Shared International Types (SIT) or M. bovis spoligotypes (SB), while we also identified seven new SB profiles (SB2695 to SB2701). Twenty-eight of the SB profiles were new to Algeria. Our data suggest zoonotic transmission in Sétif, where significantly more TB was observed among cattle (20%) compared to the slaughterhouses from the three other regions (5.4%–7.3%) (p < 0.0001), with the isolation of the same M. bovis genotypes from TB patients. The present study showed a high genetic diversity of MTBC isolated from human and cattle in Northern Algeria. Even though relatively small in terms of numbers, our data suggest the zoonotic transmission of TB from cattle to humans, suggesting the need for stronger eradication strategies for bovine TB.

MDR M. tuberculosis outbreak clone in Eswatini missed by Xpert has elevated bedaquiline resistance dated to the pre-treatment era

BackgroundMultidrug-resistant (MDR) Mycobacterium tuberculosis complex strains not detected by commercial molecular drug susceptibility testing (mDST) assays due to the RpoB I491F resistance mutation are threatening the control of MDR tuberculosis (MDR-TB) in Eswatini.MethodsWe investigate the evolution and spread of MDR strains in Eswatini with a focus on bedaquiline (BDQ) and clofazimine (CFZ) resistance using whole-genome sequencing in two collections ((1) national drug resistance survey, 2009–2010; (2) MDR strains from the Nhlangano region, 2014–2017).ResultsMDR strains in collection 1 had a high cluster rate (95%, 117/123 MDR strains) with 55% grouped into the two largest clusters (gCL3, n = 28; gCL10, n = 40). All gCL10 isolates, which likely emerged around 1993 (95% highest posterior density 1987–1998), carried the mutation RpoB I491F that is missed by commercial mDST assays. In addition, 21 (53%) gCL10 isolates shared a Rv0678 M146T mutation that correlated with elevated minimum inhibitory concentrations (MICs) to BDQ and CFZ compared to wild type isolates. gCL10 isolates with the Rv0678 M146T mutation were also detected in collection 2.ConclusionThe high clustering rate suggests that transmission has been driving the MDR-TB epidemic in Eswatini for three decades. The presence of MDR strains in Eswatini that are not detected by commercial mDST assays and have elevated MICs to BDQ and CFZ potentially jeopardizes the successful implementation of new MDR-TB treatment guidelines. Measures to limit the spread of these outbreak isolates need to be implemented urgently.

Genetic diversity of the Mycobacterium tuberculosis complex strains from newly diagnosed tuberculosis patients in Northwest Ethiopia reveals a predominance of East-African-Indian and Euro-American lineages

ObjectivesThis study described the population structure of M. tuberculosis complex (MTBc) strains among patients with pulmonary or lymph node tuberculosis (TB) in Northwest Ethiopia and tested the performance of culture isolation and MPT64-based speciation for Lineage 7 (L7). MethodsPatients were recruited between April 2017 and June 2019 in North Gondar, Ethiopia. The MPT64 assay was used to confirm MTBc, and spoligotyping was used to characterize mycobacterial lineages. Line probe assay (LPA) was used to detect resistance to rifampicin and isoniazid. ResultsAmong 274 MTBc genotyped isolates, there were five MTBc lineages: L1–L4 and L7 were identified, with predominant East-African-Indian (L3) (53.6%) and Euro-American (L4) (40.1%) strains, and low prevalence (2.6%) of Ethiopia L7. The genotypes were similarly distributed between pulmonary and lymph node TB, and all lineages were equally isolated by culture and recognized as MTBc by the MPT64 assay. Additionally, LPA showed that 259 (94.5%) MTBc were susceptible to both rifampicin and isoniazid, and one (0.4%) was multi-drug resistant (resistant to both rifampicin and isoniazid). ConclusionThese findings show that TB in North Gondar, Ethiopia, is mainly caused by L3 and L4 strains, with low rates of L7, confirmed as MTBc by MPT64 assay and with limited resistance to rifampicin and isoniazid.

A pilot study on the genetic diversity of Mycobacterium tuberculosis complex strains from tuberculosis patients in the Littoral region of Cameroon

BackgroundThe re-emergence of tuberculosis (TB) worldwide, compounded by multi-drug resistance (MDR) of the causative agents constitutes a major challenge to the management of the disease. Rapid diagnosis and accurate strain identification are pivotal to the control of the disease. This pilot study investigated the genetic diversity of Mycobacterium tuberculosis complex (MTBC) strains from TB patients in the Littoral region of Cameroon as well as their resistance to rifampicin (RIF). Patients and methodsThis was a cross sectional hospital-based study carried out between January and December 2017 and including 158 isolates from sputum smear positive individuals [105 (66.5%) males and 53 (33.5%) females]. Sputum samples were tested using Xpert MTB/RIF, followed by culture on Lowenstein–Jensen medium. Isolates were further subjected to molecular characterization using IS6110 typing, deletion analysis and spoligotyping. ResultsThirteen (8.8%) of the 147 isolates with susceptibility results available were resistant to RIF. Drug resistance occurred in 5/50 (10%) female compared to 8/97 (8.2%) male (OR, 0.81; 0.25–2.62; p = 0.764), and there was no significant difference across the age ranges (p = 0.448). On the other hand, RIF resistance was associated (OR, 0.18, 95%CI, 0.05–0.69; p = 0.023) with previously treated patients [(4/14 (28.6%)] compared to new ones [9/133 (6.8%)]. The 150 identified lineages included among others 54 (36%) Cameroon, 18 (12%) UgandaI, 32 (21.3%) Haarlem, 17 (11.3%) Ghana, 9(6%) West African 1, 7(4.7%) Delhi/CAS, 4 (2.7%) LAM and 3 (2%) UgandaII. Of the 150 isolates, the major cluster was the Cameroon SIT 61, with 43(28.7%) isolates. Six (35.3%) of the 17 UgandaI sub-lineage were RIF resistant (OR, 9.58; 95%CI, 2.74–33.55, p = 0.001). ConclusionThe cosmopolitan Littoral region presents with a wide Mycobacterium tuberculosis (MTB) strains diversity and the UgandaI sub-lineage likely associated with RIF resistance. Understanding the spread of this clade through surveillance will enhance TB control in the region.

Use of a whole genome sequencing-based approach for Mycobacterium tuberculosis surveillance in Europe in 2017-2019: an ECDC pilot study.

Whole genome sequencing (WGS) can be used for molecular typing and characterisation of Mycobacterium tuberculosis complex (MTBC) strains. We evaluated the systematic use of a WGS-based approach for MTBC surveillance involving all European Union/European Economic Area (EU/EEA) countries and highlight the challenges and lessons learnt to be considered for the future development of a WGS-based surveillance system.WGS and epidemiological data of patients with rifampicin-resistant (RR) and multidrug-resistant (MDR) tuberculosis (TB) were collected from EU/EEA countries between January 2017 and December 2019. WGS-based genetic relatedness analysis was performed using a standardised approach including both core genome multilocus sequence typing (cgMLST) and single nucleotide polymorphism (SNP)-based calculation of distances on all WGS data that fulfilled minimum quality criteria to ensure data comparability.A total of 2218 RR/MDR-MTBC isolates were collected from 25 countries. Among these, 56 cross-border clusters with increased likelihood of recent transmission (≤5 SNPs distance) comprising 316 RR/MDR-MTBC isolates were identified. The cross-border clusters included between two and 30 resistant isolates from two to six countries, demonstrating different RR/MDR-TB transmission patterns in Western and Eastern EU countries.This pilot study shows that a WGS-based surveillance system is not only feasible but can efficiently elucidate the dynamics of in-country and cross-border RR/MDR-TB transmission across EU/EEA countries. Lessons learnt from this study highlight that the establishment of an EU/EEA centralised WGS-based surveillance system for TB will require strengthening of national integrated systems performing prospective WGS surveillance and the development of clear procedures to facilitate international collaboration for the investigation of cross-border clusters.

Typing and classification of non-tuberculous mycobacteria isolates

Background: There are a large and growing number of non-tuberculous mycobacteria (NTM) species that have been isolated, identified, and described in the literature, yet there are many clinical isolates which are not assignable to known species even when the genome has been sequenced. Additionally, a recent manuscript has proposed the reclassification of the Mycobacterium genus into five distinct genera. Methods: We describe using a community standard fast average nucleotide identity (ANI) approximation method, MASH, for classifying NTM genomes by comparison to a resource of type strain genomes and proxy genomes. We evaluate the genus reclassification proposal in light of our ANI, MLST, and pan-genome work. Results: We describe here a sequencing study of hundreds of clinical NTM isolates. To aid in characterizing these isolates we defined a multi-locus sequence typing (MLST) schema for NTMs which can differentiate strains at the species and subspecies level using eight ribosomal protein genes. We determined and deposited the allele profiles for 2,802 NTM and Mycobacterium tuberculosis complex strains in PubMLST. Conclusions: The MLST schema and our pan-genome analysis of Mycobacteria can help inform the design of marker-gene diagnostics. The ANI comparisons likewise can assist in the classification of unknown genomes, even from previously unknown species.

Detection of low-frequency resistance-mediating SNPs in next-generation sequencing data of Mycobacterium tuberculosis complex strains with binoSNP

Accurate drug resistance detection is key for guiding effective tuberculosis treatment. While genotypic resistance can be rapidly detected by molecular methods, their application is challenged by mixed mycobacterial populations comprising both susceptible and resistant cells (heteroresistance). For this, next-generation sequencing (NGS) based approaches promise the determination of variants even at low frequencies. However, accurate methods for a valid detection of low-frequency variants in NGS data are currently lacking. To tackle this problem, we developed the variant detection tool binoSNP which allows the determination of low-frequency single nucleotide polymorphisms (SNPs) in NGS datasets from Mycobacterium tuberculosis complex (MTBC) strains. By taking a reference-mapped file as input, binoSNP evaluates each genomic position of interest using a binomial test procedure. binoSNP was validated using in-silico, in-vitro, and serial patient isolates datasets comprising varying genomic coverage depths (100-500×) and SNP allele frequencies (1-30%). Overall, the detection limit for low-frequency SNPs depends on the combination of coverage depth and allele frequency of the resistance-associated mutation. binoSNP allows for valid detection of resistance associated SNPs at a 1% frequency with a coverage ≥400×. In conclusion, binoSNP provides a valid approach to detect low-frequency resistance-mediating SNPs in NGS data from clinical MTBC strains. It can be implemented in automated, end-user friendly analysis tools for NGS data and is a step forward towards individualized TB therapy.

Live attenuated TB vaccines representing the three modern Mycobacterium tuberculosis lineages reveal that the Euro–American genetic background confers optimal vaccine potential

BackgroundHuman tuberculosis (TB) is caused by a plethora of Mycobacterium tuberculosis complex (MTBC) strains belonging to seven phylogenetic branches. Lineages 2, 3 and 4 are considered “modern” branches of the MTBC responsible for the majority of worldwide TB. Since the current BCG vaccine confers variable protection against pulmonary TB, new candidates are investigated. MTBVAC is the unique live attenuated vaccine based on M. tuberculosis in human clinical trials. MethodsMTBVAC was originally constructed by unmarked phoP and fadD26 deletions in a clinical isolate belonging to L4. Here we construct new vaccines based on isogenic gene deletions in clinical isolates of the L2 and L3 modern lineages. These three vaccine candidates were characterized at molecular level and also in animal experiments of protection and safety. FindingsSafety studies in immunocompromised mice showed that MTBVAC-L2 was less attenuated than BCG Pasteur, while the original MTBVAC was found even more attenuated than BCG and MTBVAC-L3 showed an intermediate phenotype. The three MTBVAC candidates showed similar or superior protection compared to BCG in immunocompetent mice vaccinated with each MTBVAC candidate and challenged with three representative strains of the modern lineages. InterpretationMTBVAC vaccines, based on double phoP and fadD26 deletions, protect against TB independently of the phylogenetic linage used as template strain for their construction. Nevertheless, lineage L4 confers the best safety profile. FundingEuropean Commission (TBVAC2020, H2020-PHC-643381), Spanish Ministry of Science (RTI2018-097625-B-I00), Instituto de Salud Carlos III (PI18/0336), Gobierno de Aragón/Fondo Social Europeo and the French National Research Council (ANR-10-LABX-62-IBEID, ANR-16-CE35-0009, ANR-16-CE15-0003).

Typing and classification of non-tuberculous mycobacteria isolates

Background: There are a large and growing number of non-tuberculous mycobacteria (NTM) species that have been isolated, identified, and described in the literature, yet there are many clinical isolates which are not assignable to known species even when the genome has been sequenced. Additionally, a recent manuscript has proposed the reclassification of the Mycobacterium genus into five distinct genera. Methods: We describe using a fast average nucleotide identity (ANI) approximation method, MASH, for classifying NTM genomes by comparison to a resource of type strain genomes and proxy genomes. We evaluate the genus reclassification proposal in light of our ANI, MLST, and pan-genome work. Results: We describe here a sequencing study of hundreds of clinical NTM isolates. To aid in characterizing these isolates we defined a multi-locus sequence typing (MLST) schema for NTMs which can differentiate strains at the species and subspecies level using eight ribosomal protein genes. We determined and deposited the allele profiles for 2,802 NTM and Mycobacterium tuberculosis complex strains in PubMLST. Conclusions: The MLST schema and our pan-genome analysis of Mycobacteria can help inform the design of marker-gene diagnostics. The ANI comparisons likewise can assist in the classification of unknown genomes, even from previously unknown species.

Mycobacterium tuberculosis Complex Lineage 3 as Causative Agent of Pulmonary Tuberculosis, Eastern Sudan1.

Pathogen-based factors associated with tuberculosis (TB) in eastern Sudan are not well defined. We investigated genetic diversity, drug resistance, and possible transmission clusters of Mycobacterium tuberculosis complex (MTBC) strains by using a genomic epidemiology approach. We collected 383 sputum specimens at 3 hospitals in 2014 and 2016 from patients with symptoms suggestive of TB; of these, 171 grew MTBC strains. Whole-genome sequencing could be performed on 166 MTBC strains; phylogenetic classification revealed that most (73.4%; n = 122) belonged to lineage 3 (L3). Genome-based cluster analysis showed that 76 strains (45.9%) were grouped into 29 molecular clusters, comprising 2–8 strains/patients. Of the strains investigated, 9.0% (15/166) were multidrug resistant (MDR); 10 MDR MTBC strains were linked to 1 large MDR transmission network. Our findings indicate that L3 strains are the main causative agent of TB in eastern Sudan; MDR TB is caused mainly by transmission of MDR L3 strains.

Molecular methods in diagnostics of post-BCG vaccine adverse events.

IntroductionBacillus Calmette-Guérin (BCG) is the only tuberculosis vaccine available and although it has been routinely used for more than 80 years, its protective effect varies depending on the age and the form of tuberculosis. Due to the close analogy between the vaccine strain and other species of the Mycobacterium tuberculosis complex, molecular methods are recommended for differential diagnosis of post-BCG complications. The aim of the study was to assess usefulness of molecular methods in diagnosis of post-BCG vaccine adverse events (VAEs).Material and methodsM. tuberculosis complex strains obtained in 2011-2017 from 68 ill children were subjected to molecular analysis.ResultsMolecular analysis of 68 strains showed 100% agreement between the results in the GenoType MTBC method and the multiplex PCR method. For the strains isolated from 45 patients with suspected VAE, M. bovis BCG was obtained, whereas the strains isolated from the remaining 23 children were identified as M. tuberculosis. The analysis confirmed the close relationship between the result of identification and the type of material as well as the patient’s age.ConclusionsThe use of genetic methods enables quick and detailed diagnostics of infections caused by M. bovis BCG, which allows for the confirmation or exclusion of VAE.

Development and application of affordable SNP typing approaches to genotype Mycobacterium tuberculosis complex strains in low and high burden countries

The Mycobacterium tuberculosis complex (MTBC) comprises the species that causes tuberculosis (TB) which affects 10 million people every year. A robust classification of species, lineages, and sub-lineages is important to explore associations with drug resistance, epidemiological patterns or clinical outcomes. We present a rapid and easy-to-follow methodology to classify clinical TB samples into the main MTBC clades. Approaches are based on the identification of lineage and sub-lineage diagnostic SNP using a real-time PCR high resolution melting assay and classic Sanger sequencing from low-concentrated, low quality DNA. Thus, suitable for implementation in middle and low-income countries. Once we validated our molecular procedures, we characterized a total of 491 biological samples from human and cattle hosts, representing countries with different TB burden. Overall, we managed to genotype ~95% of all samples despite coming from unpurified and low-concentrated DNA. Our approach also allowed us to detect zoonotic cases in eight human samples from Nigeria. To conclude, the molecular techniques we have developed, are accurate, discriminative and reproducible. Furthermore, it costs less than other classic typing methods, resulting in an affordable alternative method in TB laboratories.

Evaluation of the resistance patterns of Mycobacterium tuberculosis complex strains to antituberculous drugs

Aim: To determine the resistance profile of Mycobacterium tuberculosis complex (MTBC) strains to first-line antituberculous drugs. Methods: A total of 138 patients with MTBC growth from 2008-2018 were evaluated retrospectively. The Ehrlich-Ziehl-Neelsen (EZN) staining method was used for direct smear preparations, the BACTEC MGIT 460 TB system the Lowenstein-Jensen medium for culture planting and the BACTEC NAP test for the diagnosis of MTBC. Susceptibility tests were performed using the BACTEC MGIT 460 TB system with the streptomycin, isoniazid, rifampicin and ethambutol (SIRE) kit in accordance with the manufacturer's recommendations. Results: Of the total 138 tuberculosis (TB) cases, 44 (31.9%) were female and 94 (68.1%) were male. MTBC was most frequently isolated from pulmonary specimens (90.6%). Acid-resistant bacilli (ARB) positivity was detected in 88 (63.8%) samples by EZN staining for culture-positive samples. In our study, without considering single or multiple drug resistance (MDR), total resistance rates in MTBC strains were determined for, isoniazid (INH), rifampicin (RIF), ethambutol (EMB), and streptomycin (SM); 10.1%, 4.3%, 2.9%, and 12.3% respectively. While the susceptibility to all drugs was 82.6%, multiple drug–resistant tuberculosis (MDR-TB) was 2.9%. Conclusion: These results are important since they are the first data reported from our province regarding the determination of the resistance profile to anti-TB drugs. Resistance rates in our study were very close to the 2016 data average of the Ministry of Health of Turkey. Determination of TB resistance profiles, as well as proper and regular treatment, will contribute to the control of MDR-TB.