A. O. Mariandyshev

I. V. Tarasova

P. I. Eliseev

Russian Journal of Infection and Immunity

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5672018, vol. 8, no. 4 6. Tuberculosis and Mycobacteriafrom the same time period with the same genotype were included in the phylogenomic analysis. The genomic vari-ants identified by WGS were used for phylogenomic infer-ence, drug resistance prediction and to determine genomic distances between isolates.WGS analysis revealed unexpected genomic diversity within the seemingly homogenous IS6110 cluster of M. tu-berculosis isolates. Despite the IS6110 RFLP based uni-formity, at least six non-time dependent sub-clusters and several orphan-isolates were evident from the WGS-based phylogeny and genomic comparisons. Sub-clusters gained drug resistance conferring mutations (beyond MDR) on multiple occasions and M. tuberculosis isolates from surrounding suburbs were observed throughout the phy-logeny.IS6110 RFLP typing underestimated the complexity of this 23-year outbreak. This study suggests that there is continuous circulation and reintroduction of this M. tu-berculosis cluster in the community setting. Even with the advent of the WGS-era, confirming direct epidemio-logical links or outbreak directionality remains a challenge in high TB burden, low-income settings.6.13 doi: 10.15789/2220-7619-2018-4-6.13A 15-YEAR SPATIOTEMPORAL ANALYSIS OF MYCOBACTERIUM TUBERCULOSIS LINEAGES 1 AND 2 IN CHIANG RAI, THAILANDA. Disratthakit1, P. Palittapongarnpim2,3, P. Ajawatanawong2, N. Smittipat3, S. Mahasirimongkol1, R. Miyahara4, H. Yanai5,6, N. Yamada7, S. Nedsuwan8, W. Imasanguan8, P. Kantipong8, B. Chaiyasirinroje5, S. Bupachat5, P. Ananpradit5, W. Ruengchai2, T. Juthayothin3, J. Phelan9, J. Parkhill10, T.G. Clark9, M.L. Hibberd9, K. Tokunaga41Department of Medical Sciences, Ministry of Public Health, Thailand; 2Department of Microbiology, Faculty of Science, Mahidol University, Thailand; 3National Centre for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Thailand; 4Department of Human Genetics, Graduate School of Medicine, the University of Tokyo, Japan; 5TB-HIV Research Foundation, Thailand; 6Fukujuji Hospital, Japan Antituberculosis Association (JATA), Japan; 7Research Institute of Tuberculosis, JATA, Japan; 8Chiangrai Prachanukroh Hospital, Ministry of Public Health, Thailand; 9London School of Hygiene and Tropical Medicine, United Kingdom; 10Welcome Trust Sanger Institute, Hinxton, United KingdomChiang Rai is the northernmost province of Thailand with high burden of tuberculosis (TB) and high TB death rate. Chiang Rai consists of various ethnic groups in three different geographic areas including (1) the bordering area in the northern and northeastern, (2) the central area, and (3) the outlying districts in the southeastern, southern and southwestern.We aimed to assess the spatial and temporal distribu-tion of Mycobacterium tuberculosis (MTB) lineages in the three different areas over 15 years.Whole genome sequence (WGS) data was used to clas-sify the genotypes of 1497 MTB using lineage-specific sin-gle nucleotide polymorphisms (SNPs). The spatiotempo-ral distribution of MTB lineage was analyzed in the three different areas during early 2000s (2002–2006), late 2000s (2007–2011) and 2010s (2014–2018). Stoddart and Taylor’s index (G) was calculated to determine genotypic diversity of MTB lineage in the different settings.In 2000s, lineage 2 (East Asian) was a highly predom-inant genotype (45%) in Chiang Rai followed by lineage 1 (Indo-oceanic) (41%). In 2010s, lineage 1 became the most dominant genotype (51%) replacing lineage 2 (35%). The overall change in predominant lineage from lineage 2 to lineage 1 was caused by a dramatic increase propor-tion of lineage 1 in the central area (from 44 to 53%) and in the outlying districts (from 39 to 59%). In the bordering area, a combined impact of increasing distribution of line-age 1 (from 32 to 40%) and other lineages (from 18 to 24%) was an additional cause of changing predominant lineage. The Stoddart and Tayloy’s analysis of genotypic diversi-ty showed that the central area had the highest diversity of MTB lineage (G = 4.14±0.46) followed by the border-ing area (G = 3.67±0.76) and the outlying districts (G = 3.63±0.62).Our study combining genotypic and space-time ana-lysis has revealed a dynamic population changes in MTB lineage over 15-year period in Chiang Rai. Further studi-es on social determinants and patient’s demographic data in the different geographic areas have the potential to pro-vide effective TB control in the different setting.6.14 doi: 10.15789/2220-7619-2018-4-6.14MOLECULAR-GENETIC METHODS OF DETECTION OF TUBERCULOSIS AND ITS DRUG RESISTANCE IN ARKHANGELSK REGION IN 2017P.I. Eliseev1, I.V. Tarasova2, A.O. Mariandyshev11Northern State Medical University, Arkhangelsk, Russia; 2Arkhangelsk Regional Antituberculosis Dispensary, Arkhangelsk, RussiaBurden of tuberculosis (TB) is decreasing in Arkhan-gelsk region in northwestern Russia with incidence declin-ing from 45.9/100 000 in 2012 to 21.6/100 000 in 2017 in ci-vil society (excluding penitentiary society). Introduction of molecular-genetic tests of detection of TB and its drug resistance (DR), including multidrug-resistant (MDR) and extensively drug resistant (XDR) TB, is one of the key com-ponents of regional TB program. It plays an important role in improvement of diagnostics and management of TB pa-tients in the region.The objective was to evaluate performance of molecu-lar-genetic tests used for detection of TB and its DR among patients with TB registered in civil society in Arkhangelsk region in 2017.Line probe assay (LPA) (Hain Lifescience, Germany) and GeneXpert MTB/RIF (Cepheid, USA) were used for detection of M. tuberculosis (MTB) and its DR along-side conventional sputum smear microscopy (SM) and cul-ture. All patients were initially tested with SM followed by LPA (Genotype MTBDRplus) if result of SM was positive or GeneXpert if result was negative using the same sample. In case of DR to isoniazid and/or rifampicin, Genotype MTBDRsl was performed to detect additional DR to fluo-roquinolones and injectables, including XDR. In cases suggestive of nontuberculous mycobacteria (NTM), SM or culture positive with negative results of LPA or GeneXpert for MBT, identification was performed using Genotype Mycobacterium CM/AS.Total of 214 “new cases” and 28 “relapses” of TB were registered in Arkhangelsk region in 2017. MTB was detect-ed in 160 (74.8%) out of 214 “new cases” and all of them were tested for DR using molecular-genetic tests. 53 pa-tients (31.1%) had MDR-TB, among them 3 patients had additional DR to injectables and 2 patients had XDR-TB. Among 28 “relapses” MTB was detected in 24 (85.7%) patients. 13 patients (54.2%) had MDR-TB, among them in 1 patient additional DR to injectables and in 2 patients to fluoroquinolones was detected. NTM associated disease was diagnosed in 4 patients (2 — M. avium, 1 — M. gor-donae, 1 — M. interjectum).568Russian Journal of Infection and ImmunityAbstracts of International conferenceAll TB patients in Arkhangelsk region were tested with molecular-genetic tests before the treatment ena-bling quicker diagnostics and earlier treatment initiation. Early diagnosis ensures proper treatment regimen for pa-tients with TB and NTM diseases. As a result manage-ment of TB patients is improved leading to better treat-ment outcomes and subsequently reduced TB transmis-sion in the region.6.15 doi: 10.15789/2220-7619-2018-4-6.15DETECTION OF EXTRACELLULAR MYCOBACTERIUM TUBERCULOSIS SMALL RNAsM.V. Fursov, T.I. Kombarova, I.A. Dyatlov, V.D. PotapovState Research Center for Applied Microbiology and Biotechnology, Obolensk, RussiaAccording to WHO, tuberculosis infection is one of the top ten deadly infections in the world, and about one-fourth of the human population is a carrier of latent tuberculosis infection (LTBI) without manifestation of disease symp-toms. The LTBI associated with the conversion of M. tu-berculosis bacilli to a dormant, metabolically inactive state; however, the molecular mechanisms of this change is not well studied. In many researches the small RNAs (sRNAs) was proposed as regulators of these processes. The aim of the study was detection of sRNA transcripts in cultur-al supernatant of M. tuberculosis strain H37Rv and into the blood serum of mice (C57Bl) infected with LTBI.Mycobacterial cells were grown in Middlebrook 7H9 containing 10% ADC supplement at 37°C and harvested at different growth phase for use. The culture of M. tuber-culosis was centrifuged at 6000g for 20 min at 4°C. The su-pernatant was filtered through 0.22 μm filters to remove the remaining bacteria. Bacterial total RNA was extracted from M. tuberculosis cultural supernatant by ExtractRNA reagent (Evrogen, Moscow, Russia), followed by diges-tion with Turbo DNase-free kit (Ambion, Austin, TX, USA) to remove contaminating DNA. cDNA was syn-thesized with 1.5 μg of total RNA by M-MLV (Evrogen, Moscow, Russia) transcriptase and random hexadeoxy-nucleotides according to the manufacture’s instruction. The quantitative RT-PCR (qPCR) was carried out with the qPCRmix-HS SYBR (Evrogen, Moscow, Russia) and the CFX-96 real-time PCR detection system (BioRad, USA). All primers used in this study were designed using VectorNTI 11 (Invirogen, USA) and GeneRunner software (http://www.generunner.net) and synthesized at Evrogen (Moscow, Russia). LTBI C57Bl mouse model was designed as previously described (Shramko et al., 2010). The certain sRNA transcripts have been detected in M. tu-berculosis culture supernatant at different growth phases (exponential phase, stationary phase and late-stationary phase) and into the blood serum of mice infected with LTBI. Obtained data allow us to propose M. tuberculosis sRNAs as new markers for LTBI diagnostic in the future.6.16 doi: 10.15789/2220-7619-2018-4-6.16TB PORTALS PROGRAM: DATA-DRIVEN MULTI-NATIONAL CONSORTIUM AGAINST DRUG-RESISTANT TUBERCULOSISA.E. Gabrielian, A. RosenthalOffice of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockvill, MD, USATB Portals Program (TBPP) is an international ini-tiative that is founded, developed and steered by doctors, researchers, IT specialists and radiologists to combat drug-resistant tuberculosis (DR-TB). The efforts of hospi-tals and biomedical research institutes from ten countries on three continents are organized and supported by TBPP to actively establish and grow an integrated open-access network of innovative tools and data from real patient cases of TB (tbportals.nid.nih.gov). The TB Portals collect, ana-lyze, standardize, and present anonymized clinical, labo-ratory, and socioeconomic data, full bacterial genomes, and radiological data (CXR and CT).The TBPP database currently has more than 1300 published (22 250 total cases), 75% of which are MDR or XDR-TB. Clinicians supply and validate all patient data. Once validated, the data become published with open access status in accordance with NIH FAIR principles. Importantly, both original and derived (expert-based and computational annotations) data remain patient-centric, i.e. linked to a unique patient identifier. This cornerstone principle enables users to define and analyze cohorts of pa-tients, augmenting OMICS studies with diverse clinical in-formation.To date the database contains 730 published (1300 to-tal) fully sequenced and annotated Mycobacterium tubercu-losis genomes associated with the patient case record. We will highlight several TBPP projects studying 1) genomic signatures for TB relapse and reinfection, and 2) compara-tive analysis of M. tuberculosis strains isolated from sputum vs. surgically removed parts of lungs.TBPP assists doctors and researchers in testing and re-fining their hypotheses with friendly and powerful tools. Starting from genomics and the molecular basis of drug resistance, we will demonstrate how our online tools en-able anyone to simultaneously look at clinical, microbio-logical and radiological evidences in order to (1) search for genomic clues for inconsistencies in existing DR-TB diagnostics and to (2) study common and countries-spe-cific evolutionary patterns of M. tuberculosis.6.17 doi: 10.15789/2220-7619-2018-4-6.17SIMPLIFYING NGS APPROACHES TO OPTIMIZE TRACING OF TRANSBORDER SPREAD OF MYCOBACTERIUM TUBERCULOSISD. Garcia de ViedmaClinical Microbiology and Infectious diseases Department, Gregorio Marañón University Hospital, Madrid, Spain; CIBER Enfermedades Respiratorias CIBERES, Madrid, SpainMolecular epidemiology, and more recently genomic epidemiology, based on whole genome sequencing (WGS), improve our understanding about the transmission dy-namics of Mycobacterium tuberculosis in a population. However, in many countries, including many of those with a high burden of TB, systematic genomic epidemiology cannot be implemented. Trying to find a solution to this situation, we propose an alternative line of progression, which tries to conciliate the discriminatory power of WGS with the speed, low cost and simplicity of PCR-based approaches. The cost of this shortcut is that it sacrifices the complete knowledge of all the transmission clusters in a population, because it needs to focus on surveying the strains that deserve special attention because they are more actively transmitted, or correspond to high-risk MDR or XDR strains. This short-cut strategy has proved to be efficient to survey actively transmitted strains, to fast track outbreak-strains, to update the presence of high-risk strains in a population or to give an urgent answer to pub-lic-health alerts, such as to rule out secondary cases due to the importation of XDR-TB cases. More recently, we are integrating this strategy to optimize the characteriza-

MOLECULAR-GENETIC METHODS OF DETECTION OF TUBERCULOSIS AND ITS DRUG RESISTANCE IN ARKHANGELSK REGION IN 2017

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MOLECULAR-GENETIC METHODS OF DETECTION OF TUBERCULOSIS AND ITS DRUG RESISTANCE IN ARKHANGELSK REGION IN 2017

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