A. Dimitrova

H. Najdenski

M. Bonovska

T. Savova-Lalkovska

V. Valcheva

Russian Journal of Infection and Immunity

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5832018, vol. 8, no. 4 6. Tuberculosis and Mycobacterialates. Mutations in rrl and rrs genes were not detected. AST for 4 MAM isolates confirmed that inducible resistance is not present even with erm(41) T28 mutation. AST for 3 MAA with erm(41) C28 polymorphism showed MIC valu-es below 2 mg/L which is interpreted by CLSI guidelines as sensitive strain. AST showed that MIC values for amikacin are between 8–16 mg/L interpreted as sensitive and con-cordant with molecular analysis.In Slovenia, for macrolide and aminoglycoside resist-ance, phenotypic and genotypic results of Mycobacterium abscessus complex are concordant. Prevalent subspecies is MAA where high percentage of strains have inducible macrolide resistance. No other unknown genetic muta-tion was present in our isolates that can cause inducible macrolide resistance which is important for treatment patients with CF, where clarithromycin is first drug of choice.6.54 doi: 10.15789/2220-7619-2018-4-6.54MOLECULAR FEATURES OF MYCOBACTERIUM TUBERCULOSIS ISOLATES FROM PATIENTS LIVING IN CLOSED CITY IN THE URAL REGION, RUSSIAT.V. Umpeleva1, N.I. Eremeeva1, K.V. Belousova1, D.V. Vakhrusheva1, A.A. Vyazovaya21National Medical Research Center of Tuberculosis and Infectious Diseases, Ekaterinburg, Russia; 2St. Petersburg Pasteur Institute, St. Petersburg, RussiaNovouralsk is a closed town in the Sverdlovsk region, Middle Ural area in Russia, with a total population of 81 500 and travel and residency restrictions. We aimed to identify the molecular-epidemiological features of M. tuber culosis circulating in Novouralsk under these specific conditions.A total of 87 M. tuberculosis clinical isolates ob-tained between 2013 and 2016 from TB patients living in Novouralsk town were analyzed. According to clinical data, 34 (39.1%) of TB patients were HIV-infected. 53 (60.9%) of TB cases were newly diagnosed. Using real-time PCR we divided M. tuberculosis clinical isolates into Beijing/non-Beijing genetic groups. Beijing genotype vari-ant B0/W148 was detected by multiplex PCR assay. VNTR loci MIRU26 and QUB26 were used for subtyping Beijing strains. Spoligotyping was used for further subtyping non-Beijing isolates. Drug susceptibility testing for first and second line drugs was performed by absolute consen-tration method.Genotyping identified the predominance of the Beijing genotype isolates (75.5%), among new TB cas-es, that is almost 20% higher than the average for the Ural region (p < 0.05). 52.8% isolates belonged to vari-ant Beijing B0/W148. The majority of Beijing isolates — 35 (40.2%) had seven copies in MIRU26 and QUB-26 loci. Nine (10.3%) Beijing B0/W148 isolates had 2 copies in QUB26 locus that was unusual for this genetic cluster; six patients from this group had TB/HIV co-infection. Seven (8.0%) of non-Beijing isolates belonged to SIT35 spoligotype (Ural family). 20.7% of patient had prison history and 72.2% of them were infected with B0/W148 genotype. The MDR prevalence rate was higher than in Sverdlovsk region (66% vs 43.9%, p < 0.05) and MDR status was associated with the Beijing B0/W148 genotype (94% and 6% of its isolates were MDR and polyresistant, respectively).Epidemiological situation with TB in Novo uralsk is char-acterized by high level of TB/HIV co-infection, predomi-nance of Beijing B0/W148 isolates, which is an underlying reason of high level of MDR-TB.6.55 doi: 10.15789/2220-7619-2018-4-6.55PREVALENCE AND DIVERSITY OF NONTUBERCULOUS MYCOBACTERIA IN DIFFERENT REGIONS OF THE RUSSIAN FEDERATIONV. Ustinova1,2, T. Smirnova1, E. Larionova1, I. Andrievskaya1, E. Kiseleva1, S. Andreevskaya1, L. Chernousova11Central Tuberculosis Research Institute, Moscow, Russia; 2LLC Syntol, Moscow, RussiaToday, more attention is being paid worldwide to the nontuberculous mycobacteria (NTM) infections due to their increase in various regions of the world. The prev-alence of different NTM species depends on the geograph-ical location. The prevalence and distribution of the NTM species in Russian Federation have not been sufficiently studied to date .The objective of this study was to demonstrate the diversi-ty of NTM species isolated from patients in different regions of the Russian Federation. NTM were isolated from solid and liquid media from patients with suspected tuberculosis/mycobacteriosis in the period from July, 2013 to June, 2017 and identified using GenoType Mycobacterium CM/AS as-say (Hain Lifescience, Germany) and real-time PCR assay co-developed with Syntol LLC (Moscow, Russia) . Seventeen NTM species were identified in 1400 cultures from 876 pa-tients. Exact species was identified for isolates from 840/876 (95.89%) patients. The prevalence of slowly growing NTM was 76.5% (643/840). The most common species was M. avium (223/846, 25.5%). Also the high incidence rate (in descend-ing order) has been shown for M.gordonae (115/846, 13.12%), M. lentiflavum (113/846, 12.9%), M. intracellulare (80, 9.13%), M. fortuitum (83/846, 9.47%), M. kansasii (62/846, 7.1%), M. abscessus (52/846, 5.9%) and M. xenopi (31/846, 3.53%). 16 of 17 identified species were detected in Moscow region. This may be due to a large number of cases analyzed in this region in comparison with other regions. It was shown that NTM species distribution in Central Federal District, European part of Privolzhsky Federal District and Kaliningrad (Northwest Russia) was similar to European countries: МАС 33–39%, M. gordonae — 10–20% and M. fortuitum — 5–13%. The spe-cies distribution of NTM for neighboring Syktyvkar and Perm (North of European Russia) was similar and was cha-racterized by high rates of M. fortuitum infection and low rates of M. avium infection. NTM, isolated from the border city of Khanty-Mansiysk were also characterized by low occur-rence of M. avium and prevalence of M. gordonae.To conclude, during the study period, 1400 samples col-lected from 876 patients in 6 federal districts were analyz-ed. The greatest species diversity — 16 NTM species — was shown for Moscow region. In most of the regions anayz-ed, slow-growing NTMs prevailed and the most abundant were species belonging to MAC complex. However, even on a relatively small number of observations, some regional features can be observed.6.56 doi: 10.15789/2220-7619-2018-4-6.56MOLECULAR CHARACTERIZATION OF MYCOBACTERIUM BOVIS ISOLATES FROM CATTLE IN BULGARIAV. Valcheva1, T. Savova-Lalkovska2, A. Dimitrova2, H. Najdenski1, M. Bonovska11The Stephan Angeloff Institute of Microbiology, BAS, Sofia, Bulgaria; 2National Diagnostic and Research Veterinary Medical Institute, Sofia, BulgariaThe observation of the population dynamics, diver-sity of genotypes and dissemination of Mycobacterium bovis strains in Bulgaria and neighboring countries play 584Russian Journal of Infection and ImmunityAbstracts of International conferencean important role in the modern epidemiological investi-gations of tuberculosis in animals at the regional and in-ternational level. Bovine tuberculosis represents a signi-ficant economic burden to the agriculture of the affected countries. From 2000 to 2015 the disease shows cyclicity in private farms in different regions of Bulgaria. This study is a first molecular investigation of animal tuber-culosis in the veterinary medicine in country. The mac-roscopic and microscopic observation of 35 diagnos-tic materials from slaughtered cattle, received in the National Reference Laboratory of animal tuberculosis were studied with the three molecular methods: RD4-PCR, spoligotyping and MIRU-VNTR. In 27 of the examined lymph nodes we found specific lesions for bo-vine tuberculosis. The findings were confirmed bacte-riologically and by conventional PCR. To differentiate M. bovis from other M. tuberculosis complex subtypes, we used primers f lanking specific deletion (RD4) in the ge-nome of M. bovis and obtained the 446 bp DNA pro duct. The spoligotyping subdivided the strains into 3 spoligo-types shared by two to 20 strains. Further molecular in-vestigations of M. bovis strains are needed to characte rize the genetic diversity and population structure of M. bovis strains isolated from cattle in Bulgaria. New information will be added to the global database in the field of molec-ular epidemiology of the prevalence of M. bovis strains in the cattle population in Bulgaria, which will allow comparative analysis with data from the Balkan region and Europe.Acknowledgements: This work was supported by the grant DN16/12 of the National Science Fund, Bulgaria.6.57 doi: 10.15789/2220-7619-2018-4-6.57INTERNATIONAL VALIDATION OF ANALYSIS PIPELINES FOR WHOLE GENOME SEQUENCING DATA OF MYCOBACTERIUM TUBERCULOSIS ISOLATESD. van Soolingen1, R. Jajou1, T. Kohl2, T. Walker3, S. Niemann2, H. de Neeling1, R. Anthony11National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands; 2German Center for Infection Research, Borstel Site, Borstel, Germany; 3Nuffield Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United KingdomThe aim of this multicenter study was to validate and compare different pipelines used for analysis of the Whole Genome Sequencing (WGS) data of Mycobacterium tuber-culosis isolates.All 535 M. tuberculosis isolates of culture positive cas-es in the Netherlands in 2016, were subjected to WGS, in addition to the routine application of VNTR typing. Transmission suggested on basis of identical VNTR pro-files of cases in 2016 was further investigated by munici-pal health services and 41 epi-links were traced. Fastq.gz files of all 535 samples were analysed in four different WGS pipelines to facilitate international compari-son: 1) SNP-based method at the RIVM/Bilthoven/The Netherlands; 2) SNP-based method at Oxford Uni-versity/UK; 3) SNP-based method and 4) cgMLST at Borstel/Germany.In all pipelines, shorter than 12 SNP distances between the 41 epi-linked cases was observed. One epi-linked pair revealed a higher genetic distance of 27 SNPs in the Bilthoven pipeline, due to poor sequence quality resulting in low coverage. In general, the genetic distances between isolates of the epi-linked cases were smaller in the Oxford and Borstel pipelines (0–3 SNPs), than in the Bilthoven pipeline (1–11 SNPs). All pipelines clustered roughly the same cases, more isolates without identified epi-links were clustered in the Oxford (n = 34) and both Borstel pipelines (n = 32 in the SNP pipeline and n = 39 in the cgMLST) than the Bilthoven pipeline (n = 29).Also, some cases not clustered by VNTR were clustered by WGS. Patient characteristics revealed that in some of these pairs of cases an epi-link, missed by VNTR typ-ing, was likely.Several differences were observed among the pipe-lines with regard to the version of reference genome used, software used for mapping and SNP calling, (repetitive) regions excluded in the analysis, the minimum number of reads to support SNPs, and the minimum allele fre-quencies. The RIVM pipeline was adapted in the light of these results to function more in line with other interna-tional laboratories pipelines, facilitating the comparability of results.International standardization on all these variables is necessary, and subsequently on the SNP cut-off to be applied to WGS clustering, to allow international-labo-ratory comparison of WGS data and reliable investigation of cross-border transmission.6.58 doi: 10.15789/2220-7619-2018-4-6.58RNA-BASED DRUG SUSCEPTIBILITY TESTING OF MYCOBACTERIUM TUBERCULOSISA. Van den Bossche1, R. Bhattacharyya2, J.Y. Coppee3, L. Rigouts4, A. Baulard5, A. Vodolazkaia6, D. Hung2, V. Mathys1, P.J. Ceyssens11Division of Bacterial Diseases, Sciensano, Brussels, Belgium; 2Harvard Medical School, Boston, United States; 3Institut Pasteur, Paris, France; 4Institute of Tropical Medicine, Antwerp, Belgium; 5Center for Infection and Immunity of Lille, Institut Pasteur de Lille, France; 6Laboratory of Medical Microbiology, Sciensano, Brussels, BelgiumMultidrug resistant tuberculosis (MDR-TB) is one of the major WHO health concerns. One of the challen-ges that hampers the effective response to MDR-TB is the long turnaround time of phenotypic Drug Susceptibility Testing (DST) of Mycobacterium tuberculosis. To counter this, new fast and sensitive DNA-based methods were suc-cessfully introduced over the last years. However, these (a) are based on the knowledge on resistance mutations, (b) do not distinguish living from dead cells, (c) ignore all intrin-sic resistance mechanisms, and (d) ignore the influence of compensatory mutations.We introduce a next-generation diagnostic test based on quantification of drug-specific RNA biomarkers. The basic principle is that a brief antibiotic exposure trig-gers specific transcriptional responses in susceptible, but not in resistant, microbes within a few hours. This has the advantage that long culture-dependent steps are avoid-ed, yet the resistance phenotype is detected independent of the specific cause of resistance.First, the global transcriptional response of two M. tu-berculosis strains to 10 anti-TB drugs was determined using RNAtaq-Seq. A set of highly responsive genes was selected for each drug and RNA-targeting probes were de-signed.Next, the RNA-based DST was developed in 96 well format. In short, 200 μl of a positively flagged MGITTM (BD) culture is spiked with a drug, while a replicate is in-cubated in absence of the drug. Multiplex mRNA quanti-fication is performed directly on crude cell lysates using a combination of the bead-based MagPixTM (Luminex) and QuantigeneTM Plex (Thermo Fisher) technology. 

MOLECULAR CHARACTERIZATION OF MYCOBACTERIUM BOVIS ISOLATES FROM CATTLE IN BULGARIA

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