Scientific Program including Abstracts

Transcription

th
36
Annual Congress
of the European Society
of Mycobacteriology
28th June – 01st July 2015
Riga, Latvia
Scientific Program
including Abstracts
1
CONTENT
Welcome Message ................................................ 4
Congress Organization ......................................... 5
Sponsors / Exhibition ........................................... 6
Scientific Program . ................................................8
List of Lectures . .................................................. 13
Lectures (L) ..................................................................... 13
Guest Lectures (GL) ....................................................... 13
Gertrud Meissner Award ................................................ 13
List of Oral Presentations (OP) .......................... 14
List of Poster Presentations (P) ......................... 18
Abstracts of Lectures ......................................... 28
Impressum
Publisher
Agency KONSENS Ltd.
Stockumer Straße 30
59368 Werne
Germany
Phone: +49 23 89 / 52 75 0
Homepage: www.agentur-konsens.de
Editor
Prof. Dr. Stefan Niemann
Research Center Borstel
Parkallee 1-40
23845 Borstel
Germany
Lectures (L) ..................................................................... 28
Guest Lectures (GL) ....................................................... 30
Gertrud Meissner Award ................................................ 33
Abstracts of Oral Presentations ........................ 34
Abstracts of Poster Presentations .................... 52
Author Index ........................................................ 94
Design & Layout
Agency KONSENS Ltd.
Print
Lonnemann GmbH
Ludgeristraße 13
59379 Selm
Germany
ISBN: 978-3-00-049915-9
2
3
Welcome message
CONGRESS ORGANIZATION
Dear friends,
SCIENTIFIC ORGANIZATION
It is a great honor to announce that the 36th annual Congress of the European Society of
Mycobacteriology will take place in the very centre of Europe – Riga, the capital of Latvia,
from June 28th to July 1st, 2015.
Riga is more than 800 years old, blending a medieval centre and a modern city. Ancient,
but at the same time youthful, multicultural and European, today’s Riga surprises visitors
with its diverse and rich cultural life.
The old legend tells that Riga is a city that “will never be completed” and if completed it will
sink into the waters of the river Daugava. The same incompleteness applies to our knowledge about mycobacteria and human coexistence and this urge to know more and better
understand has driven ESM for many years.
We believe that during the congress in Riga you will be immersed into the newest scientific achievements, fruitful discussions and a rich and enjoyable social life ranging from old
traditions to a contemporary urban style.
Ģirts Šķenders, Head of Mycobacteriology Department,
Centre of Tuberculosis and Lung Diseases
Riga East University Hospital, Stopiņu novads, Latvia
Come to Riga to be inspired!
Ģirts Šķenders
STEERING COMMITEE
President Prof. Dr. Stefan Niemann (Borstel, Germany)
Vice-president Prof. Dr. phil II Gabriela Pfyffer von Altishofen
(Luzern, Switzerland)
Treasurer Nalin Rastogi (Pointe-A-Pitre, Guadeloupe, France)
Secretary Suzana David (Lisbon, Portugal)
Members MD PhD Daniela Maria Cirillo (Milano, Italy)
Dr. Sven Hoffner (Solna, Sweden)
Ģirts Šķenders (Stopiņu novads, Latvia)
LOCAL ORGANIZING AGENCY
Agency KONSENS Ltd.
Germany
4
5
SPONSORS / ExhIBITION
Exhibition map
We express our gratitude to all who supported and helped the organization of the 36th edition
of ESM congress:
Platin Medal Sponsor
Hain Lifescience GmbH
www.hain-lifescience.com
booth number: 1
Exhibition map 1st floor
Silver Medal Sponsor
BD Diagnostics – Diagnostic Systems
www.bd.com
booth number: 2
Alpha-Tec Systems Inc.
www.alphatecsystems.com
booth number: 3
Beamedex
www.beamedex.com
booth number: 5
AID – Autoimmun Diagnostika GmbH
http://aid-diagnostika.com/
booth number: 6
Board room
RIGA
Poster session
Wardrobe
Foyer
Coffee break
Applied Maths Nv
www.applied-maths.com
booth number: 4
Genoscreen
www.genoscreen.com
booth number: 4
VILNIUS
WC
Other Sponsor
Congress
office
Foyer
WC
6
1
PARIS II
PARIS I
Conference hall
Exhibition area
and Coffee break
2
3
4
5
Alere Technologies GmbH
www.alere-technologies.com
6
7
SCIENTIFIC PROGRAM
Sunday, 28.06.2015
12:00
Registration
14:00-16:30 ESM – Symposium:
Next Generation Sequencing of M. tuberculosis –Digging in the deep
Chairs: Daniela Maria Cirillo, Stefan Niemann
Lecture 1: Data complexity and standardization
Stefan Niemann
Lecture 2: A global initiative for a resistance SNP Encyclopedia
Daniela Maria Cirillo
Lecture 3: Whole-genome sequence data predicts drug-susceptibility and
resistance – a large scale approach
Timothy Walker
Lecture 4: The future of Mycobacterium tuberculosis sequencing: direct
from clinical Samples
Dave Engelthaler
16:30-17:00
Symposia organized by Industry
16:30-17:00
Symposium: HAIN
16:30-16:45
Antibiotic resistance of nontuberculous Mycobacteria
Emmanuelle Cambau and Faiza Mougari
16:45-17:00
Direct detection of Mycobacteria by the GenoType CM
Gaby E. Pfyffer von Altishofen
17:00-17:30
Coffee break in the foyer and visit of the exhibition
17:30-18:00
Shuttle Bus from Bellevue Park Hotel to Splendid Palace
18:00-18:25
Opening session:
• Congress President: Girts Skenders
• President of the ESM: Stefan Niemann
18:25-18:30
Travel Grant Awards
18:30-19:15
Gertrud Meissner Award Lecture
Chair: Stefan Niemann
HOW CAN DIAGNOSTIC WHOLE-GENOME SEQUENCING FOR TB BECOME SUSTAINABLE?
Claudio Köser (UK)
19:15-19:45
Gardner Middlebrook Award Ceremony
Chairs: Gaby E. Pfyffer von Altishofen and Salman Siddiqi
Lecture of the laureate
19:45 Welcome reception
22:00
Shuttle bus back to Bellevue Park Hotel
Monday, 29.06.2015
Morning
9:00-10:45
Global perspective of TB diagnostics
Chairs: Daniela Maria Cirillo, Hanna Soini
9:00-9:45
Guest lecture 1: New developments in diagnostics
Catharina Boehme (Switzerland)
9:45-10:05
DNA AMPLIFICATION-BASED ASSAYS FOR RAPID DIAGNOSIS OF TUBERCULOSIS IN
MOROCCO AND DETECTION OF MUTATIONS ASSOCIATED WITH RIFAMPICIN RESISTANCE
IN MYCOBACTERIUM TUBERCULOSIS (OP 1)
El Mehdi Bentaleb, Mohammed ABID, Saaïd Amzazi, Hassan Ait Benhassou, Hassan Sefrioui
10:05-10:25 Evaluation of Fluorotype MTB for the detection of Mycobacterium
tuberculosis complex DNA in paraffin-embedded biopsies (OP 2)
Zaira Moure, Josep Castellvi, Adrian Sánchez, Maria Concepción Marina,
Maria Teresa Tortola Fernandez
10:25-10:45 A multicenter study on the new version of the genotype MTBDRsl assay for
the detection of resistance to fluoroquinolones and second line
injectable drugs (OP 3)
Elisa Tagliani, Paolo Miotto, Andrea Maurizio Cabibbe, Emanuele Borroni, Juan Carlos Toro,
Mikael Mansjö, Sven Hoffner, Doris Hillemann, Aksana Zalutskaya, Alena M. Skrahina,
10:45-11:15
11:15-13:15
Global perspective of TB diagnostics
Chairs: Catharina Boehme, Girts Skenders
11:15-12:00
Guest lecture 2: GeneXpert rolL-out in South Africa: experience and impact
Wendy Stevens (South Africa)
12:00-12:20 Tailored genotyping tools based on allele-specific PCRs targeting
Mycobacterium tuberculosis strain-specific SNPs obtained from whole
genome sequencing data (OP 4)
Laura Pérez-Lago, Miguel Martínez Lirola, Iñaki Comas, Marta Herranz, Santiago Izco,
María Jesús Ruiz Serrano, Paula López Roa, Juan Carlos Momo, Lucía Biyé Ondó,
Juan Eyene, Emilio Bouza, Darío García de Viedma
12:20-12:40 PhyResSE: Web-tool for antibiotic resistance and lineage discrimination
based on whole genome sequencing data of Mycobacterium tuberculosis
complex strains (OP 5)
Viola Schleusener, Silke Feuerriegel, Patrick Beckert, Thomas Kohl, Paolo Miotto,
Andrea M. Cabibbe, Daniela Maria Cirillo, Stefan Niemann, Kurt Fellenberg
12:40-13:00
The value of microscopic-observation drug susceptibility (MODS) assay in
the diagnosis of tuberculosis and detection of multidrug resistance (OP 6)
Deniz Sertel Şelale, Meltem Uzun
13:00-14:00
Lunch in the foyer and visit of the exhibition
Afternoon
14:00-15:00
Poster session
15:00-16:30
Drug resistance of M. tuberculosis
Chairs: Patricia Del Portillo, Timothy Walker
15:00-15:45 Guest lecture 3: Understanding the MDR-TB in Eastern Europe: Whole genome
evolution of clinical M. tuberculosis isolates
Matthias Merker (Germany)
8
9
15:45-16:05
Molecular characteristics of PZA resistant strains from Tbilisi, Republic
of Georgia, a high burden MDR-TB setting (OP 7)
Sarah Sengstake, Indra Bergval, Nino Bablishvili, Nestani Tukvadze, Jessica De Beer,
Rusudan Aspindzelashvili, Dick van Soolingen, Richard Anthony
16:05-16:25 The role of MDR TB in emergence of retreatment tuberculosis cases (OP 8)
Anda Nodieva, Inta Jansone, Ilva Pole, Girts Skenders, Matiss Bauskenieks, Ilze Morozova,
Iveta Ozere, Vaira Leimane, Viesturs Baumanis, Renate Ranka
16:30-17:00
17:00-18:30
Drug resistance of M.tuberculosis
Chairs: Anandi Martin, Igor Mokrousov
17:00-17:45
Guest lecture 4: Optimizing TB drug trials
Michael Hölscher (Germany)
17:45-18:05
Pyrazinamide resistance in Mycobacterium tuberculosis fails to bite? (OP 9)
Richard Anthony, Alice den Hertog, Sarah Sengstake
18:05-18:25
The role of the Beijing and H4/Ural genotypes in the epidemiology and
treatment outcomes of multidrug-resistant tuberculosis (OP 10)
Valeriu Crudu, Elena Romancenco, Ecaterina Noroc, Liliana Domente, Sofia Alexandru,
Dumitru Chesov, Viorel Soltan, Stefan Niemann, Christoph Lange, M. Merker, Richard Garfein,
Timothy Rodwell, Antonino Catanzaro, Constantin Rimis
18:30
Shuttle bus from Bellevue Park Hotel to the Open Air museum
23:00
Shuttle bus back to Bellevue Park Hotel
Tuesday, 30.06.2015
Morning
9:00-11:00
TB Epidemiology I
Chairs: Leen Rigouts, Dick van Soolingen
9:00-9:45
Guest lecture 5: TB in children
Marc Nicol (South Africa)
9:45-10:05
Ural family of Mycobacterium tuberculosis: not sleeping beast anymore?
(OP 11)
Igor Mokrousov
12:00-12:20 Insights from molecular typing into the rapidly evolving epidemiological
landscape of tuberculosis in Sicily (OP14)
Celestino Bonura, Michel Gomgnimbou, Guislaine Refrégier, Christophe Sola, Caterina Mammina
12:20-12:40
Complex genomic diversity of Mycobacterium tuberculosis in Ethiopia
(OP 15)
Iñaki Comas, Sebastien Gagneux, David Stucki, Douglas Young, Abraham Aseffa, Stefan Berg
12:40-13:00 Comparative Intradermal Tuberculin Test in Dairy Cattle in the North of
Ecuador and Risk Factors Associated with Bovine Tuberculosis in animal
and humans (OP 16)
Freddy Proano-Perez, Lenin Ron-Garrido, Ricardo Benitez-Capistros, Francoise Portaels,
Leen Rigouts
13:00-14:00
Lunch in the foyer and visit of the exhibition
Afternoon
14:00-15:00
Poster session
15:00-15:30
15:30-18:00
Special Topic “TB elimination”
Chairs: Gaby Pfyffer, Sven Hoffner
15:30-16:15 Guest lecture 7: Can we reach the WHO End TB Global TB targets? A modelling
analysis for South Africa, China and India
Richard White (United Kingdom)
16:15-16:35 What have studies on the protein expression of Mycobacterium
tuberculosis brought us? (OP 17)
Jeroen de Keijzer
16:35-16:55 Identification of mutations affecting candidate genes potentially involved
in phenotypic resistance to bedaquiline and delamanid in M. Tuberculosis
(OP 18)
Simone Battaglia, Andrea Maurizio Cabibbe, Elisa Schena, Emanuele Borroni, Paolo Miotto,
Daniela Cirillo
16:55-17:15
Detection of pyrazinamide heteroresistance in M. Tuberculosis (OP19)
Jim Werngren, Mikaela Glader, Sven Hoffner
17:15-18:00
General assembly
18:30 Optional: Sight-Seeing Tour through Riga
10:05-10:20 Map African TB - Population structure and evolution of Mycobacterium
tuberculosis complex in Africa (OP 12)
Patrick Beckert, Ellen Bruske, Florian Gehre, Elisabeth Sanchez-Padilla, Véronique N. Penlap Beng,
Abraham Alabi, Bertrand Lell, Thomas A. Kohl, Francine Ntoumi, Matthias Frank, Maryline Bonnet,
Bouke C. de Jong, Andrea Rachow, Michael Hölscher, Stefan Niemann
20:00
Party at the Bellevue Park Hotel
10:20-10:40 Challenges in setting-up molecular diagnostic tools for tuberculosis
drug resistance detection and epidemiology studies in low-income
countries (OP 13)
Voahangy Rasolofo, Marie Sylvianne Rabodoarivelo, Noël Ratovonirina,
Solohery Razafimahatratra, Juan Carlos Palomino, Christophe Sola, Fanjasoa Rakotomanana,
Anandi Martin
Wednesday, 01.07.2015
10:45-11:15
11:15-13:00
TB Epidemiology II
Chairs: Sebastien Gagneux, Stefan Niemann
11.15-12.00
Guest lecture 6: TB transmission in Western European settings
Thimothy Walker (United Kingdom)
10
Morning
9:00-11:00Host – Pathogen Interaction
Chairs: Mark Nicol, Richard Anthony
9:00-9:45
Guest lecture 8: Linking M. tuberculosis complex population structure with
pathobiology
Sebastien Gagneux (Switzerland)
9:45-10:05
The impact of lipids in the global genetic expression of Mycobacterium
tuberculosis during its adaptation to dormancy (OP 20)
Diana Angelica Aguilar Ayala, Dieter De Coninck, Dieter Deforce, Filip Van Nieuwerburgh,
Juan Carlos Palomino, Peter Vandamme, Jorge González Y Merchand, Anandi Martin
11
10:05-10:25
Ms1, a novel sRNA interacting with the RNAP core in mycobacteria (OP 24)
Libor Krásný, Jarmila Hnilicová, Michaela Šiková, Martina Janoušková, Jiří Pospíšil
10:25-10:45 CXCL9, CXCL10, and IL6 cytokines as potential biomarkers of treatment
response in pulmonary TB patients (OP 22)
Vladyslav Nikolayevskyy, Yanina Balabanova, Irina Kontsevaya, Olga Ignatyeva, Edita Pimkina,
Girts Skenders, Francis Drobniewski
LIST OF LECTURES
L1
Data complexity and standardization
Stefan Niemann
L2
A global initiative for a resistance SNP Encyclopedia
10:45-11:05
Evidence for Mycobacterium avium ssp. paratuberculosis specific zinc
transport systems (OP 23)
Elke Eckelt, Ralph Goethe, Jochen Meens, Michael Jarek
11:05-11:30
11:30-13:45
Environmental mycobacteria
Chairs: Suzana David, Darío García de Viedma
11:30-12:15
Guest lecture 9: NTM - update 2015
Enrico Tortoli (Italy)
L4
The future of Mycobacterium tuberculosis sequencing: direct from clinical samples
Dave Engelthaler
12:15-12:35
Preliminary in silico-based insights on the functionality of CRISPR-Cas in
M. tu bercul osis complex (OP 21)
Guislaine Refrégier, Matthieu Petrou, Christophe Sola
LIST OF GUEST LECTURES
12:35-12:55
Incidence and diversity of nontuberculous mycobacteria in Moscow region
(OP 25)
Maria Alvarez Figueroa, Valentina Barilo, Anastasiya Prokopenko
GL1
New developments in diagnostics
Catharina Boehme
12:55-13:15 First genotyping study of clinical and environmental Mycobacterium
ulcerans isolates from French Guiana reveals unprecedented genetic
diversity (OP 26)
Yann Reynaud, Julie Millet, David Couvin, Aaron Morris, Rodolphe Gozlan,
Jean-François Guéguan, Nalin Rastogi, Pierre Couppié , Eric Legrand
13:15-13:25
Analysis of Mycobacterium abscessus Genetic Variability provided by
14-locus Variable-Number Tandem-Repeat in Patients with Cystic Fibrosis
(OP 27)
Alberto Trovato, Daniela Maria Cirillo, Rossella Baldan, Enrico Tortoli, Stefan Niemann,
Giovanni Taccetti, Silvia Campana, Lisa Cariani, Paola Maria Rancoita, Thomas Kohl,
Tullia Simonetti
13:30- 13:45 Poster Price awards
13:45-14:00
Closing remarks
L3
Whole-genome sequence data predicts drug-susceptibility and resistance – a large
scale approach
Timothy Walker
GL2
GeneXpert roLl-out in South Africa: experience and impact
Wendy Stevens
GL3
Understanding the MDR-TB in Eastern Europe: Whole genome evolution of clinical M.
tuberculosis isolates
Matthias Merker
GL4
Optimizing TB drug trials
Michael Hölscher
GL5
Guest lecture 5: TB in children
Marc Nicol
GL6
TB transmission in Western European settings
Timothy Walker
GL7
Can we reach the WHO End TB Global TB targets? A modelling analysis for South
Africa, China and India
Richard White
GL8
Linking M. tuberculosis complex population structure with pathobiology
Sebastien Gagneux
GL9
NTM – update 2015
Enrico Tortoli
Gertrud meissner award
How can diagnostic whole-genome sequencing for TB become sustainable?
Claudio Köser
12
13
LIST OF ORAL PRESENTATIONS (OP)
GLOBAL PERSPECTIVE OF TB DIAGNOSTICS
OP 1
DNA AMPLIFICATION-BASED ASSAYS FOR RAPID DIAGNOSIS OF
TuBERCuLOSIS IN MOROCCO AND DETECTION OF MuTATIONS ASSOCIATED
WITh RIFAMPICIN RESISTANCE IN MYCOBACTERIuM TuBERCuLOSIS
El Mehdi Bentaleb, Mohammed Abid, Saaïd Amzazi, Hassan Ait Benhassou, Hassan
Sefrioui
OP 2
EVALuATION OF FLuOROTYPE MTB FOR ThE DETECTION OF MYCOBACTERIuM
TuBERCuLOSIS COMPLEx DNA IN PARAFFIN-EMBEDDED BIOPSIES
zaira Moure, josep Castellvi, Adrian Sánchez, Maria Concepción Marina, Maria teresa
tortola Fernandez
OP 3
A MuLTICENTER STuDY ON ThE NEW VERSION OF ThE GENOTYPE MTBDRsl
ASSAY FOR ThE DETECTION OF RESISTANCE TO FLuOROquINOLONES AND
SECOND LINE INjECTABLE DRuGS
Elisa tagliani, Paolo Miotto, Andrea Maurizio Cabibbe, Emanuele Borroni, juan Carlos
toro, Mikael Mansjö, Sven Hoffner, Doris Hillemann, Aksana zalutskaya, Alena M.
Skrahina, Daniela Maria Cirillo
OP 4
TAILORED GENOTYPING TOOLS BASED ON ALLELE-SPECIFIC PCRs TARGETING
MYCOBACTERIuM TuBERCuLOSIS STRAIN-SPECIFIC SNPs OBTAINED FROM
WhOLE GENOME SEquENCING DATA
Laura Pérez-Lago, Miguel Martínez Lirola, Iñaki Comas, Marta Herranz, Santiago Izco,
María jesús Ruiz Serrano, Paula López Roa, juan Carlos Momo, Lucía Biyé ondó,
juan Eyene, Emilio Bouza, Darío García de viedma
OP 5
PhyResSE: WEB -TOOL FOR ANTIBIOTIC RESISTANCE AND LINEAGE
DISCRIMINATION BASED ON WhOLE GENOME SEquENCING DATA OF
MYCOBACTERIuM TuBERCuLOSIS COMPLEx STRAINS
viola Schleusener, Silke Feuerriegel, Patrick Beckert, thomas kohl, Paolo Miotto,
Andrea M. Cabibbe, Daniela Maria Cirillo, Stefan Niemann, kurt Fellenberg
OP 6
ThE VALuE OF MICROSCOPIC-OBSERVATION DRuG SuSCEPTIBILITY (MODS)
ASSAY IN ThE DIAGNOSIS OF TuBERCuLOSIS AND DETECTION OF MuLTIDRuG
RESISTANCE
Deniz Sertel Şelale, Meltem uzun
14
DRuG RESISTANCE OF M.tuberculosis
OP 7
MOLECuLAR ChARACTERISTICS OF PZA RESISTANT STRAINS FROM TBILISI,
REPuBLIC OF GEORGIA, A hIGh BuRDEN MDR-TB SETTING
Sarah Sengstake, Indra Bergval, Nino Bablishvili, Nestani tukvadze, jessica De Beer,
Rusudan Aspindzelashvili, Dick van Soolingen, Richard Anthony
OP 8
ThE ROLE OF MDR TB IN EMERGENCE OF RETREATMENT TuBERCuLOSIS
CASES
Anda Nodieva, Inta jansone, Ilva Pole, Girts Skenders, Matiss Bauskenieks, Ilze
Morozova, Iveta ozere, vaira Leimane, viesturs Baumanis, Renate Ranka
OP 9
PYRAZINAMIDE RESISTANCE IN MYCOBACTERIuM TuBERCuLOSIS FAILS TO
BITE?
Richard Anthony, Alice den Hertog, Sarah Sengstake
OP 10
ThE ROLE OF ThE BEIjING AND h4/uRAL GENOTYPES IN ThE EPIDEMIOLOGY
AND TREATMENT OuTCOMES OF MuLTIDRuG-RESISTANT TuBERCuLOSIS
valeriu Crudu, Elena Romancenco, Ecaterina Noroc, Liliana Domente, Sofia Alexandru,
Dumitru Chesov, viorel Soltan, Stefan Niemann, Christoph Lange, M. Merker, Richard
Garfein, timothy Rodwell, Antonino Catanzaro, Constantin Rimis
TB EPIDEMIOLOGY I
OP 11
uRAL FAMILY OF MYCOBACTERIuM TuBERCuLOSIS: NOT SLEEPING BEAST
ANYMORE?
Igor Mokrousov
OP 12
MAP AFRICAN TB - POPuLATION STRuCTuRE AND EVOLuTION OF
MYCOBACTERIuM TuBERCuLOSIS COMPLEx IN AFRICA
Patrick Beckert, Ellen Bruske, Florian Gehre, Elisabeth Sanchez-Padilla, véronique N.
Penlap Beng, Abraham Alabi, Bertrand Lell, thomas A. kohl, Francine Ntoumi, Matthias
Frank, Maryline Bonnet, Bouke C. de jong, Andrea Rachow, Michael Hölscher, Stefan
Niemann
OP 13
ChALLENGES IN SETTING-uP MOLECuLAR DIAGNOSTIC TOOLS FOR
TuBERCuLOSIS DRuG RESISTANCE DETECTION AND EPIDEMIOLOGY STuDIES
IN LOW-INCOME COuNTRIES
voahangy Rasolofo, Marie Sylvianne Rabodoarivelo, Noël Ratovonirina, Solohery
Razafimahatratra, juan Carlos Palomino, Christophe Sola, Fanjasoa Rakotomanana,
Anandi Martin
15
TB EPIDEMIOLOGY II
OP 14
INSIGhTS FROM MOLECuLAR TYPING INTO ThE RAPIDLY EVOLVING
EPIDEMIOLOGICAL LANDSCAPE OF TuBERCuLOSIS IN SICILY
Celestino Bonura, Michel Gomgnimbou, Guislaine Refrégier, Christophe Sola, Caterina
Mammina
OP 15
COMPLEx GENOMIC DIVERSITY OF MYCOBACTERIuM TuBERCuLOSIS IN
EThIOPIA
Iñaki Comas, Sebastien Gagneux, David Stucki, Douglas young, Abraham Aseffa,
Stefan Berg
OP 16
COMPARATIVE INTRADERMAL TuBERCuLIN TEST IN DAIRY CATTLE IN
ThE NORTh OF ECuADOR AND RISk FACTORS ASSOCIATED WITh BOVINE
TuBERCuLOSIS IN ANIMAL AND huMANS
Freddy Proano-Perez, Lenin Ron-Garrido, Ricardo Benitez-Capistros, Francoise
Portaels, Leen Rigouts
SPECIAL TOPIC “TB ELIMINATION”
OP 17
WhAT hAVE STuDIES ON ThE PROTEIN ExPRESSION OF MYCOBACTERIuM
TuBERCuLOSIS BROuGhT uS?
jeroen de keijzer, P. de Haas, A. Mulder, j. de Beer, A. de Ru, P. van veelen, Dick van
Soolingen
OP 18
IDENTIFICATION OF MuTATIONS AFFECTING CANDIDATE GENES POTENTIALLY
INVOLVED IN PhENOTYPIC RESISTANCE TO BEDAquILINE AND DELAMANID IN
M. TuBERCuLOSIS
Simone Battaglia, Andrea Maurizio Cabibbe, Elisa Schena, Emanuele Borroni, Paolo
Miotto, Daniela Cirillo
OP 19
DETECTION OF PYRAZINAMIDE hETERORESISTANCE IN M. TuBERCuLOSIS
Jim Werngren, Mikaela Glader, Sven Hoffner
OP 21
PRELIMINARY IN SILICO-BASED INSIGhTS ON ThE FuNCTIONALITY OF CRISPRCas IN M. TuBERCuLOSIS COMPLEx
Guislaine Refrégier, Matthieu Petrou, Christophe Sola
OP 22
CxCL9, CxCL10, AND IL6 CYTOkINES AS POTENTIAL BIOMARkERS OF
TREATMENT RESPONSE IN PuLMONARY TB PATIENTS
vladyslav Nikolayevskyy, yanina Balabanova, Irina kontsevaya, olga Ignatyeva, Edita
Pimkina, Girts Skenders4, Francis Drobniewski
OP 23
EVIDENCE FOR MYCOBACTERIuM AVIuM SSP. PARATuBERCuLOSIS SPECIFIC
ZINC TRANSPORT SYSTEMS
Elke Eckelt, Ralph Goethe, jochen Meens, Michael jarek
ENVIRONMENTAL MYCOBACTERIA
OP 24
Ms1, A NOVEL sRNA INTERACTING WITh ThE RNAP CORE IN MYCOBACTERIA
Libor krásný, jarmila Hnilicová, Michaela Šiková, Martina janoušková, jiří Pospíšil
OP 25
INCIDENCE AND DIVERSITY OF NONTuBERCuLOuS MYCOBACTERIA IN
MOSCOW REGION
Maria Alvarez Figueroa, valentina Barilo, Anastasiya Prokopenko
OP 26
FIRST GENOTYPING STuDY OF CLINICAL AND ENVIRONMENTAL
MYCOBACTERIuM uLCERANS ISOLATES FROM FRENCh GuIANA REVEALS
uNPRECEDENTED GENETIC DIVERSITY
yann Reynaud, julie Millet, David Couvin, Aaron Morris, Rodolphe Gozlan, jeanFrançois Guéguan, Nalin Rastogi, Pierre Couppié , Eric Legrand
OP 27
ANALYSIS OF MYCOBACTERIuM ABSCESSuS GENETIC VARIABILITY PROVIDED
BY 14-LOCuS VARIABLE-NuMBER TANDEM-REPEAT IN PATIENTS WITh CYSTIC
FIBROSIS
Alberto Trovato, Daniela Maria Cirillo, Rossella Baldan, Enrico tortoli, Stefan Niemann,
Giovanni taccetti, Silvia Campana, Lisa Cariani, Paola Maria Rancoita, thomas kohl,
Tullia Simonetti
hOST – PAThOGEN INTERACTION
OP 20
ThE IMPACT OF LIPIDS IN ThE GLOBAL GENETIC ExPRESSION OF
MYCOBACTERIuM TuBERCuLOSIS DuRING ITS ADAPTATION TO DORMANCY
Diana Angelica Aguilar Ayala, Dieter De Coninck, Dieter Deforce, Filip van
Nieuwerburgh, juan Carlos Palomino, Peter vandamme, jorge González y Merchand,
Anandi Martin
16
17
LIST OF POSTER PRESENTATIONS (P)
P7
inhA AND katG MuTATIONS ASSOCIATED WITh ISONIAZID RESISTANCE IN A
CANADIAN PROVINCE
Sara Christianson, Meenu Sharma, David Long, Hong zhou, zhiwei Gao, Courtney
Heffernan, Richard Long, Joyce Wolfe
P8
INVASTIGATION OF PRIMARY ANTITuBERCuLOSIS DRuG RESISTANCE uSING
BACTEC MGIT 960 SYSTEM IN MYCOBACTERIuM TuBERCuLOSIS COMPLEx
STRAINS
Ilhan Afsar, yusuf Saglam, Selcuk kaya, Mustafa Demirci
P 15
PERFORMANCE OF FOuR TRANSPORT AND STORAGE SuPPORTS FOR
MOLECuLAR DETECTION OF MuLTIDRuG RESISTANT TuBERCuLOSIS
Marie Sylvianne Rabodoarivelo, Belen Imperiale, Angela Brandao, Parveen kumar,
Sarman Singh, Lucilaine Ferrazoli, Nora Morcillo, voahangy Rasolofo, juan Carlos
Palomino, Peter vandamme, Anandi Martin
P 19
IN VITRO ANTI-MYCOBACTERIAL ACTIVITY OF SELECTED MEDICINAL PLANTS
AGAINST MYCOBACTERIuM TuBERCuLOSIS AND MYCOBACTERIuM BOVIS
STRAINS
Abdella Gemechu, Mirutse Gidaqy, Gobena Ameni, Adane Worku
P 102
PREVALENCE OF BEIjING GENOTYPE AMONG MDR ISOLATES OF
MYCOBACTERIuM TuBERCuLOSIS FROM POLAND
Monika kozińska, Ewa Augustynowicz kopeć, Sylwia Brzezińska, Agnieszka
Napiórkowska, Anna zabost
P 113
MOLECuLAR INVESTIGATION OF MuTATIONS ASSOCIATED WITh RIFAMPICIN
AND ISONIAZID RESISTANCE IN CLINICAL ISOLATES OF MYCOBACTERIuM
TuBERCuLOSIS COMPLEx IN MODENA, ITALY
Giulia Fregni Serpini, Sara tagliazucchi, Francesca Frascaro, Antonella Grottola, Nadia
Nanni, Giulia Forbicini, Rita Magnani, William Gennari, Anna Fabio, Fabio Rumpianesi,
Monica Pecorari
P 114
COMPLETE GENOME SEquENCE OF ThE MuLTIDRuG-RESISTANT BEIjING-LIkE
STRAIN MYCOBACTERIuM TuBERCuLOSIS 323 uSING ThE PacBio REAL-TIME
SEquENCING PLATFORM
juan Germán Rodríguez, Camilo Pino, Andreas tauch, Martha I Murcia
18
P 129
POPuLATION STRuCTuRE OF MuLTIDRuG-RESISTANT MYCOBACTERIuM
TuBERCuLOSIS ISOLATES
Claudia Llerena Polo, Martha I Murcia, juan Germán Rodríguez, julio Guerra
P 156
GENOTYPING AuTOMATIZATION: PRELIMINARY RESuLTS ON FIRST AND
SECOND-LINE DRuG RESISTANCE DETECTION IN M. TuBERCuLOSIS COMPLEx
Harrison Magdinier Gomes, Bernice klotoe, Barbara Molina, jose Dominguez, Ali Sajid,
Maria Helena Feres Saad, Michel kiréopori Gomgnimbou, Christophe Sola
P 175
PhENOTYPIC FLuOROquINOLONE SuSCEPTIBILITY TESTING IN M.
TuBERCuLOSIS COMPARING REMA, MGIT 960 AND SOLID MEDIuM
Nele Coeck, Bouke de jong, Leen Rigouts
P 191
PhENOTYPIC AND GENOTYPIC INVESTIGATION OF MuLTIDRuG RESISTANCE
(MDR) AND ExTENSIVELY DRuG RESISTANCE (xDR) IN MYCOBACTERIuM
TuBERCuLOSIS STRAINS ISOLATED FROM CLINICAL SAMPLES IN CukuROVA
REGION, TuRkIYE
togrul Nagiyev, Emel yarar, Begum Kayar, Farhad kohansal, Gulfer yakici, taylan
Bozok, Fatih koksal
P 192
INVESTIGATION OF EPIDEMIOLOGICAL ChARACTERISTICS AND MOLECuLAR
MEChANISMS OF MOxIFLOxACIN RESISTANCE IN MYCOBACTERIuM
TuBERCuLOSIS STRAINS ISOLATED FROM PuLMONARY TuBERCuLOSIS
PATIENTS
taylan Bozok, Begum Kayar, Gulfer yakici, Mahdi Marzi, togrul Nagiyev, Emel yarar,
Firat karsli, Fatih koksal
P 193
FLuROquINOLONE hETERORESISTANCE
Leen Rigouts, Mirjam Schats, Nele Coeck, Bouke de jong
P 196
MuTATION ANALYSIS OF MYCOBACTERIAL RPOB GENES AND EVALuATING
MICS FOR RIFAMPIN IN MYCOBACTERIuM TuBERCuLOSIS STRAINS ISOLATED
FROM SPuTuM SAMPLES OF PuLMONARY TuBERCuLOSIS PATIENTS
Begum Kayar, taylan Bozok, Adnan Atilgan, Fatih koksal
P 203
INCIDENCE OF DRuG RESISTANT TuBERCuLOSIS IN RuRAL hAITI: A
DESCRIPTIVE ANALYSIS
R. justin May, Md. Siddiqur Rahman khan, Madsen Beau de Rochars, Michael
Lauzardo
P 214
ROLE OF PuTATIVE EFFLux GENE Rv0194 IN DRuG RESISTANCE IN
MYCOBACTERIuM TuBERCuLOSIS
Anshika Narang, Astha Giri, Mridula Bose, Mandira varma-Basil
19
P 215
DRuG-RESISTANT TuBERCuLOSIS IN CROATIA, 2010-2014
Mihaela obrovac, Ljiljana zmak, vera katalinic-jankovic
P 39
IN VITRO ACTIVITIES OF EDP-420 AND EDP-322 AGAINST SEVERAL
NONTuBERCuLOuS MYCOBACTERIA
Carolyn Shoen, Mary Sklaney, Michael Cynamon
P 44
RNA AND PROTEIN INTERACTING PARTNERS OF MYCOBACTERIuM SMEGMATIS
RNAP
jiří Pospíšil, jarmila Hnilicová, Martina janoušková, Libor krásný
P 46
GENE AND GENOME ANALYSIS INDICATES ThAT ThE STRAIN jS623 IS
WRONGLY CONSIDERED A MEMBER OF ThE SPECIES MYCOBACTERIuM
SMEGMATIS
Maria jesus Garcia, S. Gola
P 86
NEW GENOME SEquENCE OF A MAP ShEEP STRAIN: jIII-386 FROM GERMANY //
ANNOTATION AND COMPARISON WITh PuBLIShED MAP-S AND MAP-C STRAINS
Petra Möbius, Martin Hölzer, Marius Felder, Gabriele Nordsiek, Marco Groth, Heike
köhler, katrin Reichwald, Matthias Platzer, Manja Marz
P 96
IN VITRO ACTIVITIES OF BEDAquILINE AGAINST NON-TuBERCuLOuS
MYCOBACTERIA
Diana Angelica Aguilar Ayala, jorge Gonzalez y Merchand, koen Andries, Peter
vandamme, juan Carlos Palomino, Anandi Martin
P 120
IDENTIFICATION OF NON-TuBERCuLOuS MYCOBACTERIA CLINICAL ISOLATES
BY TANDEM DNA SEquENCING AND MALDI-TOF MS PROTEIN ANALYSIS
johana Monteserin, Roxana Paul, Margo Cnockaert, Maria Belén orandoni, Beatriz
Lopez, Palomino juan Carlos, Peter vandamme, viviana Ritacco, Anandi Martin
P 121
MASTITIS IN COWS CAuSED BY MYCOBACTERIuM FORTuITuM – A NEW
MOMENT IN DIAGNOSTICS AND TREATMENT
Silvio Spicic, Miroslav Benić Benić, Gordan kompes, vera katalinić-janković, Maja
zdelar-tuk, Irena Reil, Sanja Duvnjak, Mateja Pate, Luka Cvetnić, Željko Cvetnić
20
P 174
DECIPhERING ThE VIRuLENCE FACTORS OF ThE OPPORTuNISTIC PAThOGEN
MYCOBACTERIuM COLOMBIENSE
Monica Natalia Gonzalez Perez, Martha I Murcia, Carlos Parra-Lopez, jochen Blom,
Andreas Tauch
P 189
DETERMINATION OF DISTRIBuTION AND ANTIBIOTIC SuSCEPTIBILITY OF
NONTuBERCuLOuS MYCOBACTERIA (NTM) SPECIES IN CLINICAL SAMPLES
FROM ThE CukuROVA REGION, TuRkIYE
togrul Nagiyev, Farhad kohansal, Emel yarar, Begum Kayar, taylan Bozok, Gulfer
yakici, Fatih koksal
GLOBAL PERSPECTIVE OF TB DIAGNOSTICS
P 16
COMPARISON OF GENExPERT MTB ASSAY AND CONVENTIONAL CuLTuRE
METhODS FOR ThE DIAGNOSIS OF TuBERCuLOSIS
Ilhan Afsar, Aslı Gamze Sener, Selcuk kaya
P 32
EVALuATION OF SPEED-OLIGO DIRECT TB CASSETTE FOR DIRECT DETECTION
OF M. TuBERCuLOSIS COMPLEx FROM SPuTuM SPECIMENS
Abiy Aklilu, Abenezer Feleke, Chala Chaburte, Boja Dufera, Silvia Blanco, Pablo
Mendoza
P 65
COMPARISON OF RAPID MGIT TBC IDENTIFICATION TEST AND MYCOLIC
ACID ANALYSIS BY hIGh PERFORMANCE LIquID ChROMATOGRAPhY FOR
CONFIRMATION OF MYCOBACTERIuM TuBERCuLOSIS COMPLEx
Ilhan Afsar, yusuf Saglam, Erkan yula, Aslı Gamze Sener, Huseyin Baskin, Selcuk kaya
P 66
RELATIONAL SEquENCING TB DATA PLATFORM (ReSeqTB): GLOBAL
COLLABORATIVE EFFORT TO BuILD A CENTRALIZED RELATIONAL DATABASE
FOR INVESTIGATING ThE CORRELATIONS BETWEEN M. TuBERCuLOSIS
GENOTYPES AND DRuG RESISTANCE
Paolo Miotto, David Dolinger, timothy Rodwell, Angela Starks, Daniel johnson, Marco
Schito, Matteo zignol, Stefan Niemann, Daniela M. Cirillo
P 79
COMPARISON OF TWO NEW TRANSCRIPTION-REVERSE TRANSCRIPTION
CONCERTED REACTION METhODS (TRCR-160 M.TB and TRCR-80) FOR RAPID
DETECTION OF M. TuBERCuLOSIS COMPLEx IN RESPIRATORY SPECIMENS
Antonio Mazzarelli, Maria Grazia Paglia, Alessandro Piscini, ornella Butera, Eugenio
Bordi, Silvia D’Arezzo, Antonella vulcano, Carolina venditti, Carla Nisii, Antonino Di Caro
21
P 118
Increasing Mycobacterial growth using TiKa supplements
Tulika Munshi, Kai Hilpert, Tim Bull
P 126
Our first results with the GeneXpert MTB/RIF System®
Maria Müllerová
P 188
Application of In vivo Microbial Antigen Discovery (InMAD) for
the identification of M. tuberculosis circulating antigens in an
experimental murine model
Iris Estrada-García, Bibiana Patricia Ruiz-Sánchez, Jessica Castañeda-Casimiro,
Alejandro Francisco-Cruz, Jeanet Serafín López, Isabel Wong-Baeza, Dulce MataEspinosa, Rogelio Hernández-Pando, Sergio Estrada Parra
P 135
Detection of latent tuberculosis in HIV-infected patients from
simon bolivar and santa clara hospitals at bogotá, colombia
Martha I Murcia, Francy Johanna Pérez Llanos, Magda Beltrán, Liliana Sánchez, Carlos
Parra-López, Myriam Navarrete, Angélica Knudson, Ricardo Sánchez
P 199
Evaluation of the FluoroType® MTB Assay for use with Nondecontaminated Specimens at the Irish Mycobacteria Reference
Laboratory (IMRL)
Philomena Raftery, Margaret Fitzgibbon
P 136
Mycobacterial infections in HIV-infected individuals in bogotá,
colombia
Juan Germán Rodríguez, Martha I Murcia, Magda Beltrán, Francy Johanna Pérez
Llanos, Liliana Sánchez, Carlos Parra-López, Myriam Navarrete, Angélica Knudson,
Ricardo Sánchez
P 216
The Efficiency and Reliability of Commercial TK L Anti Tb PNB Kit for
Rapid Anti-Tuberculosis Drug Susceptibility Testing
Ismail Ceyhan, Zehra Ortarık, Resul Altınsoy
P 138
A Post Implementation Review of the Cepheid Xpert MTB RIF Assay
Version G4 for the Diagnosis of Pulmonary and Extra-Pulmonary
Tuberculosis in a Large Acute Tertiary Care Institute
Azhar Hamdan, Justine Woei Ling Peh, Yen Ee Tan, Li-Hwei Sng
P 141
Diagnosis of Mycobacterium tuberculosis from pleural fluid in
Bogota, Colombia
Martha I Murcia, Saray Ossa, Juan Germán Rodríguez
P 145
Undiagnosed Tuberculous Osteomyelitis of the tibia caused by
Mycobacterium tuberculosis complex; A Case Report
Gülnur Tarhan, Sadık Akgun, Abdülkadir Sari, Hakan Sayiner, Bülent Petik, Ismail AĞIR,
H.İbrahim Erdoğdu, Şükrü Mehmet Ertürk
P 153
“TB-SPRINT” on sputum DNA extracts from Ethiopia: first
comparison to GeneXpert and Genotype MTBDRplus
Barbara Molina-Moya, Silvia Blanco, Mulualem Agonafir, Michel Gomgnimbou, Lizania
Spinasse, Guislaine Refrégier, Daniel Datiko, Luis Cuevas, Jose Dominguez, Christophe
Sola
P 162
Comparative and computational analysis of Russian M. bovis BCG
strain
Ruslan Ludannyy, Maria Alvarez Figueroa, Diana Levi, Natalia Aleksandrova, Michail
Markelov, Vladimir Dedkov, German Shipulin
22
P 23
The sigma factor SigD of Mycobacterium tuberculosis enhances
the transcriptional expression of the septum site determining
protein in stationary phase
Nora Rios-Sarabia, Miguel Angel Ares, Jorge Gonzalez-y-Merchand
P 38
Phagocyte cells bactericide characteristics in patients with
pulmonary tuberculosis
Marina Dyakova, Olga Titarenko, Diljara Esmedlyaeva, Viatcheslav Zhuravlev
P 45
Participation of non-coding RNAs in the adaptation of
Mycobacterium tuberculosis to a lipid environment
M Ares, Maria Carmen Menendez, JG Rodriguez, AC Hernandez, J Gonzales-yMerchand, C Helguera, JR Bustos, JM Anzola, MM Zambrano, MJ Garcia, P Del Portillo
P 97
The growth rate of the strains Mycobacterium tuberculosis
Beijing isolated before and during treatment and in the time of
passages on Lowenstein-Jensen medium
Olga Manicheva, Marine Dogonadze, Natalia Melnikova, Viatcheslav Zhuravlev, Anna
Zmaznova, Anna Vyazovaya, Igor Mokrousov, Olga Narvskaya, Boris Vishnevskiy
P 109
Mycobacterium tuberculosis dormancy: a systematic study
Barbara Tizzano, Stefan Niemann
P 210
Recognition of ZnT8, proinsulin and homologous MAP peptides in
Sardinian children at risk of T1D precedes detection of classical
islet autoantibodies
Leonardo A Sechi, Magda Niegowska, Daniela Paccagnini
23
TB EPIDEMIOLOGY
P 30
GENOTYPING OF MuLTIDRuG RESISTANT MYCOBACTERIuM TuBERCuLOSIS
ISOLATES IN jALISCO MExICO
Gladys Lopez-Avalos, Martin Lopez-Rodriguez, Manuel Sandoval-Diaz, juan Carlos
villanueva-Arias, Carlos vázquez-Chacon, Ikuri Alvarez-Maya
P 51
MONITORING OF MYCOBACTERIuM BOVIS FROM FREE-RANGING WILDLIFE IN
SOuTh kOREA
yun-Ho jang, Soyoon Ryoo, yoonra jang, Narae kim, jin kyoung kim, Hang Lee, Soyoung Park, Woong-seog Song, jong-taek kim, Soo Hee Lee, jae Myung kim
P 67
MOLECuLAR SNAPShOT OF MYCOBACTERIuM TuBERCuLOSIS POPuLATION
IN REPuBLIC OF kARELIA, RuSSIAN FEDERATION
Anna vyazovaya, Natalia Solovieva, julia kononenko, Natalia Melnikova, tatiana
Sunchalina, Daria Starkova, Igor Mokrousov, viatcheslav zhuravlev, olga Narvskaya
P 84
PRACTICAL APPLICATION OF MOLECuLAR GENOTYPING OF MYCOBACTERIuM
TuBERCuLOSIS FOR ThE TuBERCuLOSIS CONTROL IN ThE COuNTRY
Iveta ozere, Inta jansone, Ilva Pole, Girts Skenders, Anda Nodieva, Anita Skangale,
olga Bobrikova, zita Lauska, Ruta Pastare, Nina Gusarevica, Margarita Baumane,
Matiss Bauskenieks, Renate Ranka, viesturs Baumanis
P 100
LABORATORY CROSS CONTAMINATION – MOLECuLAR EVIDENCE OF FALSEPOSITIVE CuLTuRES OF MYCOBACTERIuM TuBERCuLOSIS
Ewa Augustynowicz kopeć, Monika kozińska, katarzyna Wasiak, Anna Borek, Dagmara
Borkowska, Magdalena klatt
P 101
ExPLORING ThE SOCIODEMOGRAPhIC AND CLINICAL FEATuRES OF
ExTRAPuLMONARY TuBERCuLOSIS IN SAuDI ARABIA
Sahal Al-Hajoj Al-Nakhli, Bright varghese
P 142
DIRECT SPOLIGOTYPING ON DNA ExTRACTED FROM MYCOBACTERIuM
TuBERCuLOSIS POSITIVE ZIEhL-NEELSEN STAINED SLIDES FROM EThIOPIA
Barbara Molina-Moya, Silvia Blanco, Mulualem Agonafir, Michel Gomgnimbou, Lizania
Spinasse, Meissiner Gomes, Guislaine Refrégier, Daniel Datiko, Luis Cuevas, jose
Dominguez, Christophe Sola
P 149
GENETIC DIVERSITY OF MYCOBACTERIuM TuBERCuLOSIS IN NIGERIA BASED
ON SPOLIGOTYPING ON DNA ExTRACTED FROM POSITIVE ZIEhL-NEELSENSTAINED SLIDES
Barbara Molina-Moya, Silvia Blanco, Michel Gomgnimbou, Lizania Spinasse, Meissiner
Gomes, Guislaine Refrégier, Luis Cuevas, Lovett Lawson, Saddiq t. Abdurrahman,
joshua obasanya, jose Dominguez, Christophe Sola
24
P 163
CLONAL COMPLExITY IN MYCOBACTERIuM TuBERCuLOSIS IS MORE ThAN
AN ANECDOTAL FINDING: 2 ExAMPLES OF hOW IT ChALLENGES STANDARD
DIAGNOSTIC PRACTICE
Laura Pérez-Lago, Miguel Martínez Lirola, yurena Navarro, Marta Herranz, María jesús
Ruiz Serrano, Pilar Miralles, Emilio Bouza, Darío García de viedma
P 166
INTERLABORATORY EVALuATION AND OPTIMIZATION OF qIAxcel ADVANCED
SYSTEM FOR MIRu-VNTR TYPING OF MYCOBACTERIuM TuBERCuLOSIS
Alberto Trovato, Broda Agnieszka, Emanuele Borroni, vladyslav NIkolayevskyy, Francis
Drobniewski, Daniela Maria Cirillo
P 169
DISTINCT MYCOBACTERIuM TuBERCuLOSIS SPOLIGOTYPES-RELICTS OR
INVASIVE STRAINS IN LATVIA
Ilva Pole, Girts Skenders, Inta jansone, viktorija Igumnova, janis Pjalkovskis, Anda
Nodieva, Iveta ozere, vija Riekstina, Renate Ranka
P 176
INVESTIGATING MICROEVOLuTION OF M. TuBERCuLOSIS DuRING
TRANSMISSION
Anzaan Dippenaar, Margaretha de vos, Ruben van der Merwe, Gian van der Spuy, Rob
Warren, Samantha Sampson, Paul van Helden, Arnab Pain
P 179
WhOLE GENOME SEquENCE OF MYCOBACTERIuM SuRICATTAE, ThE MAIN
CAuSE OF TuBERCuLOSIS IN MEERkATS (SuRICATA SuRICATTA)
Anzaan Dippenaar, Sven Parsons, julian Drewe, Abdallah Abdallah, Ruben van der
Merwe, keith Siame, Alan Christoffels, Samantha Sampson, Nico Gey van Pittius, Paul
van Helden, Rob Warren, Arnab Pain
P 181
hIGh-ThROuGhPuT MYCOBACTERIAL INTERSPERSED REPETITIVE uNIT–
VARIABLE NuMBER TANDEM-REPEAT GENOTYPING FOR MYCOBACTERIuM
TuBERCuLOSIS EPIDEMIOLOGICAL STuDIES
Marie Gauthier, Floriane Bidault, Amandine Mosnier, Nino Bablishvili, Nestani tukvadze,
Silaphet Somphavong, Phimpha Paboriboune, oksana ocheretina, jean William Pape,
Glaucia Paranhos-Baccala, jean-Luc Berland
P 182
DETECTING PSEuDO-BEIjING M. TuBERCuLOSIS ISOLATES uSING TBMINER
Memona yasmin, jérôme Azé, Rubina tabassum, Sabira tahseen, Shahid Abbasi,
Naeem Akthar, Riwan Iqbal, Christophe Sola, Guislaine Refrégier
P 194
DECIPhERING TuBERCuLOSIS DYNAMICS IN EAST GREENLAND: GENOME
BASED MOLECuLAR EPIDEMIOLOGY OF MYCOBACTERIuM TuBERCuLOSIS IN
A hIGh INCIDENCE, LOW DIVERSITY SETTING
karen Bjorn-Mortensen, Aase Bengaard Andersen, karin Ladefoged, Bolette Soborg,
Anders koch, troels Lillebaek, Thomas Kohl, Stefan Niemann
25
P 195
COMPARISON OF IS6110, MIRu-VNTR AND SPOLIGOTYPING METhODS WhICh
ARE uSED FOR ASSOCIATING OF PhYLOGENETIC RELATIONS OF MuLTI DRuG
RESISTANT (MDR) MYCOBACTERIuM TuBERCuLOSIS ISOLATES
Gulfer yakici, Begum Kayar, taylan Bozok, togrul Nagiyev, Mahdi Marzi, Emel yarar,
Fatih koksal
P 200
SPOLIGOTYPES OF Mycobacterium tuberculosis ISOLATES FROM PATIENTS
RESIDENTS IN STATE OF PARá, BRAZIL
Emilyn Conceição, Maria Luiza Lopes, Ismari Perini Furlaneto, Harrison Gomes
Magdinier, Philip Noel Suffys, Nalin Rastogi, David Couvin, Rafael Silva Duarte, karla
valéria Batista Lima
P 204
MISCLASSIFICATION BY SPOLIGOTYPING: MuLTI-DRuG RESISTANT
MYCOBACTERIuM TuBERCuLOSIS OF ThE LATIN AMERICAN MEDITERANEAN
LINEAGE WRONGLY IDENTIFIED AS MYCOBACTERIuM PINNIPEDII (ST863)
Elis R. Dalla Costa, Sidra E. G. vasconcelos, Leonardo Esteves, Harrison M. Gomes,
Lia L. Gomes Fiocruz, Pedro E. A. Silva, joão Perdigão, Miguel viveiros, Gisela unis,
Nalin Rastogi, Maria Lucia R. Rossetti, Philip Noel Suffys
P223
CLuSTER IDENTIFICATION AND ANALYSIS OF CLINICAL MYCOBACTERIuM
TuBERCuLOSIS ISOLATES IN ThE STATE OF hAWAII
kent koster, Lishi qian, Caitlin Flores, terry Weber, Richard Brostrom, jeffrey Foster,
James Douglas
P 127
ANALYTICAL SENSITIVITY (LIMIT OF DETECTION), ANALYTICAL REACTIVITY
(INCLuSIVITY) AND ANALYTICAL SPECIFICITY (CROSS-REACTIVITY) OF
ThE BD MAx MDR-TB ASSAY FOR ThE DETECTION OF MYCOBACTERIuM
TuBERCuLOSIS COMPLEx AND RIFAMPICIN AND ISONIAZID RESISTANCE
Ira Ashman, Eliza Alexander, Ashley Anderson, kalola Andrews, kelley Bryan-McNeal,
krystal Hamlet, jackie Harris, jeffry Leitch, Stephanie Suit, Michael Porter
P 212
IDENTIFICATION OF NON-TuBERCuLOuS MYCOBACTERIA SPECIES IN ROuTINE
CLINICAL PRACTICE uSING MALDI-TOF MASS SPECTROMETRY
Eva Sodja, Nada Šorli Peranović, Helena Ribič, Manca Žolnir-Dovč
P 213
DRuG SuSCEPTIBILITY PROFILE AND MOLECuLAR TYPING OF PREDOMINANT
SINGLE NuCLEOTIDE POLYMORPhISM (SNP) CLuSTER GROuPS IN DELhI
Mandira varma-Basil, kushal Garima, Anshika Narang, Soumitesh Chakravorty, Naresh
kumar, Shraddha Podwal, Astha Giri, thierry zozio, David Couvin, Stefan Niemann,
Nalin Rastogi, David Alland, Mridula Bose
P 219
STuDYING ThE METABOLIC ACTIVITY OF MYCOBACTERIuM TuBERCuLOSIS
ThROuGh A NEW RESAZuRIN REDuCTION BASED ASSAY
Carla Silva, Joao Perdigao, Luísa jordão, Isabel Portugal
P 220
IDENTIFICATION OF ThE BEST MIRu-VNTR SET TEChNIquE FOR MDR-TB AND
xDR-TB SuRVEILLANCE IN LISBON, PORTuGAL
Carla Silva, Joao Perdigao, Luisa jordao, Isabel Portugal
P 221
DRuG RESISTANCE AND GENETIC DIVERSITY OF MYCOBACTERIuM
TuBERCuLOSIS IN LuANDA, ANGOLA: A MOLECuLAR EPIDEMIOLOGICAL
PERSPECTIVE
Joao Perdigao, Sofia Clemente, jorge Ramos, Pedro Masakidi, Diana Machado, Carla
Silva, Isabel Couto, Miguel viveiros, Nuno taveira, Isabel Portugal
26
27
ABSTRACTS OF LECTuRES (L)
L1
NExT GENERATION SEquENCING - DATA
COMPLExITY AND STANDARDIZATION
Stefan Niemann
Molecular Mycobacteriology, research center
borstel, borstel, Germany
Next Generation Sequencing (NGS) allows for rapid
analysis of nearly complete genomes of clinical
Mycobacterium tuberculosis complex (MtBC) isolates.
NGS based whole genome sequencing (WGS) analysis
goes far beyond conventional molecular tests e.g. for
drug susceptibility testing by detection of variants
in virtually all target genes involved in resistance
development (resistome analysis). Simultaneously, it
allows for high-resolution strain comparison e.g. for
outbreak analysis or even longitudinal genome based
molecular epidemiological studies. The application
of WGS for diagnostics and strains comparison has
become realistic due to the development on so-called
bench top NGS systems that can be integrated into
a normal laboratory workflow, and allow for WGS of
clinical isolates few days and at low costs.
However, the widespread use of NGS in the
mycobacterial laboratory is hampered by several
challenges. The enormous amount of data generated
requires new approaches for data analysis, data
interpretation, and standardisation of analysis
workflows. one major obstacle is the phenotypic
interpretation of the genetic data: the genotype phenotype challenge. Although some databasing
tools are already available such as tBDReaMDB,
more comprehensive and well-curated databases
are needed in order to better and more quantitatively
correlate genetic data with drug susceptibility
phenotypes. Furthermore, the datasets generated
(mainly SNP lists) are difficult to standardize and,
therefore, not yet employed for inter-laboratory
prospective surveillance.
Recently, we have developed a fully automated web
based NGS interpretation pipeline PhyResSE (https://
bioinf.fz-borstel.de/mchips/phyresse/) that allows the
delineation of phylogentic lineage and resistance
from NGS data for people untrained in bioinformatics.
It combines elaborate data processing and quality
control as befits human diagnostics with a treasure
trove of validated resistance data collected from
well-characterized samples in-house and worldwide.
Further developments are ongoing e.g. in the global
initiative of the ResSeqtB consortium
For standardization of NGS data for molecular
epidemiological applications, we developed a core
genome multi locus sequence typing (cgMLSt)
scheme for clinical MtBC isolates using the Ridom
SeqSphere+ software that transfers the genome wide
28
•
single nucleotide polymorphism (SNP) diversity into an
allele numbering system that is standardized, portable
and not computational intensive. In first evaluations,
cgMLSt allows for a meaningful epidemiological
interpretation of the WGS genotyping data. It enables
standardized WGS genotyping for epidemiological
investigations, e.g. on the regional public health office
level, and the creation of web accessible databases
for global TB surveillance with an integrated early
warning system.
L2
A GLOBAL INITIATIVE FOR A RESISTANCE SNP
ENCYCLOPEDIA
Daniela Maria Cirillo and the ReSeqTB
Consortium
San Raffaele Scientific Institute, Milan, Italy
Rapid drug susceptibility tests (RDSts) are required
to improve the management of tuberculosis (tB)
patients, guide the appropriate use of the new drugs
and new therapeutic regimens at the programmatic
level, and to prevent a further increase of drugresistant tB. the field has recognized the need for a
data-sharing platform that provides a one-stop data
source for clinically relevant genotypic and phenotypic
information on Mycobacterium tuberculosis (MtB).
Having large sets of data reviewed, validated and
made easily accessible in a user-friendly manner
will support the development of new diagnostic
tools and rules to interpret the relationship between
genetic polymorphisms and drug resistance in order
to properly manage TB patients. The Relational
Sequencing tB Data Platform, or ReSeqtB, will
fulfill this scope serving as a single repository for the
compilation, curation, and validation of existing and
newly created sequences and metadata on MtB
strains. This initiative is led by a global partnership
of academic institutions, public health agencies and
non-governmental organizations including the Critical
Path Institute, FIND, the World Health organization,
the New Diagnostics Working Group, the u.S. Centers
for Disease Control and Prevention, and the National
Institute of Allergy and Infectious Diseases and it is
financially supported by the Bill and Melinda Gates
Foundation.
the goals of ReSeqtB initiative are to provide:
• A one-stop data source for clinically relevant
and global genotypic and phenotypic information
on MtB polymorphisms associated with drug
resistance
• An improved research resource to enable
development of new diagnostic tests that can
rapidly detect drug specific resistance through
the detection of MtB genotypic markers
A foundation for developing a curated reference
resource for clinicians
Successful execution of such an extensive
database platform will require substantial, worldwide
collaboration of scientists investigating the genetic
basis for drug resistance and developers with
expertise in database design and implementation.
L3
WhOLE-GENOME SEquENCE DATA PREDICTS
DRuG-SuSCEPTIBILITY AND RESISTANCE – A
LARGE SCALE APPROACh
Timothy Walker
Department of Microbiology and infectious
Diseases, John radcliffe Hospital, oxford, uK
Diagnosing drug-resistance remains an obstacle to
the WHo’s aim of eliminating tuberculosis. Phenotypic
drug susceptibility testing (DSt) is expensive and
slow, whilst commercial genotypic assays screen
only common resistance-determining mutations
affecting a sub-set of drugs. As the price of wholegenome sequencing (WGS) technology is reducing
and as sequencing platforms are becoming more
mobile, we seek to characterise mutations predicting
drug-resistance, or consistent with susceptibility, for
all first and second-line drugs to contribute towards
a knowledgebase for a WGS-based diagnostic test.
We sequenced a training-set of 2099 Mycobacterium
tuberculosis
genomes,
characterising
each
nucleotide-position in relation to the pan-susceptible
reference genome as containing either a ‘wild-type’ or
mutant base-call. For 23 candidate-genes identified
from the drug-resistance literature, mutations were
algorithmically characterised as not conferring
resistance (‘benign’), ‘resistance-determinants’, or
‘uncharacterised’. We then assessed the capacity
of these characterisations to predict phenotypic
DST for an independent validation-set of 1552
genomes. Outside of candidate-genes we sought
mutations under similar selection pressure to those
characterised as resistance-determinants within
the candidate genes to explain residual phenotypic
resistance. 120 training-set mutations were
characterised as resistance-determining, and 772 as
‘benign’. using these mutations, 89·2% of validationset phenotypes could be predicted with a mean
92·3% sensitivity and 98·4% specificity. only 10.8%
of validation-set phenotypes could not be predicted
because uncharacterised mutations were present.
On in silico comparison, characterised resistancedeterminants had a higher sensitivity than the
mutations from three line-probe assays (85·1% vs.
81·6%). No additional resistance-determinants were
identified among mutations under selection-pressure
in non-candidate-genes. A broad catalogue of genetic
mutations enable WGS-data to be used clinically
to predict drug-resistance, drug-susceptibility, or
to identifying drug-phenotypes that cannot yet be
genetically predicted. This approach could already be
integrated into routine diagnostic workflows, phasingout phenotypic DST whilst reporting drug-resistance
earlier. The performance will improve iteratively as
more strains are sequenced.
L4
ThE FuTuRE OF MycobActeriuM
tuberculosis SEquENCING: DIRECT FROM
CLINICAL SAMPLES
David M. Engelthaler1, Rebecca E. Colman1,
jason W. Sahl1, Valeriu Crudu2, Donald
Catanzaro3, Antonino Catanzaro4, Paul keim1,5,
Ted Cohen6, Timothy C. Rodwell4
1
translational Genomics research institute, usA
2
Phthisiopneumology institute, republic of
Moldova
3
university of Arkansas, usA
4
university of california san Diego
5
Northern Arizona university
6
yale university
tabletop DNA sequencers now allow for rapid and
robust sequencing of pathogen isolates as well as
direct analysis of clinical samples. However, applying
this technology directly to complex samples, such as
sputum, currently has limitations due to the complexity
of biological samples. Barriers to progress include,
obtaining sufficient target DNA, differentiating host/
pathogen DNA, detecting genomic areas of interest,
capturing phylogenetic signal and employing the
necessary bioinformatics resources. Our group has
developed accesible tools and methodologies for
direct sequencing of clinical sputum samples which
enable us to detect Mycobacterium tuberculosis
(Mtb), produce a rapid drug susceptibility profiles,
detect heteroresistance and conduct molecular
epidemiology analyses. Methods described here
will include: A) Next Gen rapid drug suitability
testing (RDSt), an amplicon sequencing method
for generating a rapid and inexpensive DSt profile
straight from AFB positive sputum samples; B) Single
Molecule overlapping Read (SMoR) analysis –
an advanced amplicon sequencing approach for
detecting and measuring heteroresistance to 0.1%
minor resistance component; and C) Whole Genome
Focused Array SNP typing (WG-FASt), which can
place whole sample (metagenome) sequencing data
on a well-characterized phylogenetic tree. This last
approach provides a rapid method for strain typing,
and possibly contact tracing direct from a sputum
sample. We have employed these techniques on DNA
extracted >150 remnant clinical sputum samples from
Moldova. using rapid DNA sequencing with tabletop
instruments and automated analysis directly on
clinical samples, allows for this technology to move
closer to the point of care in clinics around the world.
29
ABSTRACTS OF GuEST LECTuRES (GL)
GL1
NEW DEVELOPMENTS IN DIAGNOSTICS
Catharina Boehme
GL2
GENExPERT ROLL-OuT IN SOuTh AFRICA:
ExPERIENCE AND IMPACT
Wendy Stevens
GL3
uNDERSTANDING MDR-TB IN EASTERN
EuROPE: WhOLE GENOME EVOLuTION OF
CLINICAL M. tuberculosis ISOLATES
Matthias Merker, Silke Feuerriegel, helen Cox,
Sonia Borrell, Sebastien Gagneux, Sabine
Rüsch-Gerdes, Thomas A. kohl, ulrich Nübel,
Philipp Supply, Thierry Wirth, Stefan Niemann
research center borstel, borstel, Germany
transmission of multidrug resistant (MDR, resistance to
isoniazid and rifampicin) Mycobacterium tuberculosis
complex (MtBC) strains constitutes a major threat
for global tuberculosis (tB) infection control. overall
high rates of MDR-tB among new cases in Eastern
European countries and in the Russian Federation
already point towards an ongoing transmission of
drug resistant strains. In that regard, it is unclear if we
have to cope with local MDR-tB outbreaks caused by
different, individual strain types or if we have to deal
with highly successful and widespread clones that
may have specifically adapted to MDR-tB treatment
regimens.
Beside social and economical factors the MtBC
Beijing lineage and its specific genetic background
is still under debate to contribute to an enhanced
transmissibility, high rates of drug resistances and/
or increased virulence. However, without a detailed
classification of worldwide circulating Beijing strains
clear associations between genetic variants and
pathobiological parameters is complex.
to address this questions we utilized a comprehensive
dataset of 4,987 clinical Beijing isolates originated
from 99 countries worldwide and 24-loci mycobacterial
interspersed repetitive unit – variable number tandem
repeat (MIRu-vNtR) typing. We could differentiate
seven clonal complexes, whereas only two complexes
are dominating in regions with the highest MDR-tB
incidence rates. the high clonality, i.e. extremely low
genetic diversity, was confirmed by whole genome
30
sequencing (WGS) of 110 representative isolates and
additionally pointed out new potential drug resistance
variants and possible virulence mechanisms in both
outbreak clones. An extended WGS analysis of over
1,000 Beijing outbreak strains further revealed an
ongoing transmission since at least 1995 beyond
national boarders towards the European region with
increasing rates of second-line drug resistance and
compensatory mutations.
the long-term selection of beneficial gene variants
in prominent MtBC outbreak strains needs to be
considered for future TB control programs and may
jeopardize existing treatment regimens and resistance
diagnostics due to the abundance and high diversity
of resistance and bacterial fitness determinants.
GL4
OPTIMIZING TB DRuG TRIALS
Michael hölscher
Department for infectious Diseases & tropical
Medicine, university of Munich, lMu, Munich,
Germany
This is an exciting time for tuberculosis drug
development with a number of clinical trials reporting
(REFAquIN, oFLotuB, REMoxtB). the challenge
facing drug development is illustrated by the
flouroquinolones trials to prove the promising results
in Phase 2 regimens. It is time that we review drug
development methodologies critically. The dizzying
number of potential dosages and combinations of
novel, established and repurposed agents requires
rapid pre-selection of the most promising regimens
from pre-clinical and early phase clinical trials. These
regimens must be optimized at low cost to select
the right regimen for a pivotal registration trial. A
combination of innovative animal models, early clinical
trial designs that gathers safety and DDI data as early
as possible and efficacy outcome measurements in
phase II studies that do not only look for bacillary
clearance from sputum but also for relapse is needed.
This presentation will review the different approaches
that are currently under consideration.
GL5
TB IN ChILDREN
Marc Nicol
GL6
TB TRANSMISSION IN WESTERN EuROPEAN
SETTINGS
Timothy Walker
Department of Microbiology and infectious
Diseases, John radcliffe Hospital, oxford, uK
Control of tuberculosis depends on the timely
identification and treatment of cases and the
identification of contacts. Many Western European
countries have programmes to systematically
genotype M. tuberculosis cultures, using 24-locus
MIRu-vNtR to help identify outbreaks. over the past
4 years there have been a number of different studies
demonstrating the increased resolution of wholegenome sequencing (WGS). the technology is being
piloted in England this year with a view to adoption
in 2016.
the principle advantage of WGS over MIRu-vNtR
is increased resolution for delineating the margins
of outbreaks. Additional advantages include more
detailed insights into outbreak structure with the
possibility of even illuminating the direction of
transmission, and that the same data lends itself to
the identification of species and drug susceptibility at
no extra cost. The challenges include data storage
and searching.
I will discuss the impact of WGS on our understanding
of transmission in Western Europe, using oxfordshire
and Birmingham as case studies, and will also
explore our ability to make inferences about the
direction of transmission from genetic data alone. I
will also discuss our experiences of using WGS as
a diagnostic tool in real time and show how we can
efficiently interrogate a large back-catalogue of strains
with each new sequence as it becomes available.
GL7
CAN ThE POST-2015 WhO END TB TARGETS BE
REAChED? A MuLTI-MODEL
ExERCISE FOR ChINA, INDIA AND SOuTh
AFRICA
Richard White1, houben RMGj 1, Gomez G 2,
Vassall A 1, Pillay Y 3, Wang L 4, Rade k 5, PhiriNkhoma T 6, kimerling M 7
1
london school of Hygiene and tropical
Medicine, london, uK
2
AiGHD, Amsterdam, the Netherlands
3
NDoH, Pretoria, south Africa
4
NtP, beijing, china
5
rNtcP, india
6
Action, lilongwe, Malawi
7
bill and Melinda Gates Foundation, seattle,
usA
Background
the new post-2015 global tB targets require a
50% and 75% reduction in incidence and mortality
respectively by 2025, but it is unclear how feasible
these Targets are for high-burden countries.
Methods
Eleven independently-developed dynamic transmission models of tB projected the epidemiological
impact of intervention scenarios employing existing
tB prevention and patient care tools in China, India
and South Africa. Representatives from National
tuberculosis Programmes (NtP) and the Advocacy
community provided country-specific intervention
scenarios, which included symptom screening,
active case finding and preventive therapy, enabled
by anticipated investments in health system and
social protection schemes. A combination scenario
considered the potential impact of scaling-up all
interventions simultaneously.
Findings
No single intervention scenario was sufficient
to reach WHO 2025 milestones in any country.
However, models projected that in the South Africa
NTP scenario a combination of continuous IPT for
those on ARt, increased screening for tB symptoms
at health centres and improvements in TB care could
achieve a median of 52% (range=48-60%) and 74%
(range=70-85%) reduction in incidence and mortality,
respectively. For India and China a cumulative 1.1mln
deaths and 3.2mln cases might be prevented by
2025, though the reductions from this intervention set
still fell short of the 2025 Targets in both countries.
The Advocacy scenarios illustrated the high impact of
dramatic reductions of the latent TB reservoir.
Interpretation
While major reductions in tB burden appear possible
with current tools, additional interventions, focusing
on country-specific drivers of the tB epidemic will be
needed to reach the post-2015 Global TB targets.
GL8
LINkING M. TuBERCuLOSIS COMPLEx
POPuLATION STRuCTuRE WITh
PAThOBIOLOGY
Sebastien Gagneux
Head tuberculosis research unit, Deputy Head
Dep. of Med. Parasitology & infection biol.
swiss tropical & Public Health institute, basel,
switzerland
Mycobacterium tuberculosis Lineage 4 is globally the
most important cause of human tuberculosis. However,
the factors underlying the success of this lineage are
unknown. Here we analyzed 3,366 clinical isolates
from 100 countries by combining high-throughput
genotyping with whole genome sequencing. We
show that Lineage 4 comprises four sublineages
that are geographically restricted, consistent with
a specialist phenotype, and three sublineages that
occur globally, supporting a generalist phenotype. our
data further demonstrate that European colonization
31
and migration contributed to the global spread of the
most frequent generalist sublineage of Lineage 4.
taken together, our findings indicate that the overall
success of Lineage 4 is a consequence of both the
various evolutionary strategies adopted by different
sublineages and variation in human migrations across
the continents.
GL9
NTM - uPDATE 2015
Enrico Tortoli
emerging bacterial Pathogens unit, san raffaele
Scientific Institute, Milano, Italy
Bacterial phylogeny can be reconstructed on the basis
on comparative analysis of the genetic sequences
of the single species. The 16S rRNA has been for
many years the only target of such analyses but in
the last decade other housekeeping genes have
been investigated and the phylogeny based on their
concatenated sequences has become a standard. It
is now clear that whole genomes can provide more
comprehensive data for the differentiation of species
and subspecies. Novel criteria, among which the
average nucleotide identity which relies on whole
genome sequence comparison of all conserved
genes, have been proposed, as alternative to the
poorly repeatable DNA-DNA hybridization, for the
phylogenetic definition of species. to better understand
the phylogenetic relationship between the species of
the genus Mycobacterium more than 50 type strains
were de novo sequenced using the Illumina HiSeq
platform. Following assembling and annotation
with proper software the phylogenetic analysis was
conducted with PhyloPhlAn and the pan-genome
analysis pipeline. the emerging phylogenetic three,
including also the genomes available in GenBank,
was characterized by the clear-cut distinction of slowly
and rapidly growing species with the latter being more
ancestral. The species of the Mycobacterium terrae
complex occupied an intermediate position between
rapid and slow growers. Both species located in
isolated branches and in clusters were present;
among the latter, stand the groups, or complexes,
having, as more representative species M. avium, M.
simiae, M. scrofulaceum, M. celatum, M. fortuitum,
M. chelonae, and M. smegmatis. Similarities and
differences with previous phylogenetic theories will
be object of discussion.
32
ABSTRACT OF GERTRuD MEISSNER AWARD
hOW CAN DIAGNOSTIC WhOLE-GENOME SEquENCING FOR TB BECOME SuSTAINABLE?
Claudio köser
Department of Medicine, university of cambridge,
cambridge, uK
In light of recent proof of concept studies, it is beyond a doubt that whole-genome sequencing (WGS)
of every single new culture-positive TB case will become the standard of care in well-resourced countries.
This transition will primarily be driven by the desire to
replace traditional typing techniques, such as MIRuvNtR, rather than to identify resistant strains, as resistance is relatively rare in these settings. unlike MIRu-vNtR, however, it is not sufficient to invest a set
amount of money upfront to translate WGS. Instead,
continuous public investment is required given that
WGS analysis infrastructure has to be constantly refined. As a result, the paradigm used to develop MIRuvNtR is not appropriate for WGS, which, I will argue,
calls for a fundamental change in which translational
research is currently undertaken.
33
ABSTRACTS OF ORAL PRESENTATIONS (OP)
GLOBAL PROSPECTIVE OF TB DIAGNOSTICS
OP1
DNA AMPLIFICATION-BASED ASSAYS FOR
RAPID DIAGNOSIS OF TuBERCuLOSIS IN
MOROCCO AND DETECTION OF MuTATIONS
ASSOCIATED WITh RIFAMPICIN RESISTANCE IN
El Mehdi Bentaleb1, Mohammed Abid2, Saaïd
Amzazi3, hassan Ait Benhassou4, hassan
Sefrioui4
1
Medical biotechnology center, Moroccan
Foundation for Advanced science, innovation
and research, rabat; laboratory of
biochemistry & immunology, Faculty of science,
Mohammed V university, rabat, Morocco
2
laboratoire de Génétique Mycobactérienne,
institut Pasteur, tangier, Morocco
3
laboratory of biochemistry & immunology,
Faculty of science, Mohammed V university,
rabat, Morocco
4
Medical biotechnology center, Moroccan
Foundation for Advanced science, innovation
and research, rabat, Morocco
tuberculosis (tB) is one of the deadliest infectious
diseases worldwide. It is caused by Mycobacterium
tuberculosis that infects a new person every second in
the world. Each year, about 1% of the world population
is newly infected and about 9 million people develop
the disease and approximately 2 million of them die.
In Morocco as in most developing countries where
the timely and accurate diagnosis of TB remains a
great challenge, tB constitutes a major public health
threat with 30,000 new cases each year. Furthermore,
control of TB has been complicated by the emergence
of multidrug-resistant (MDR) M. tuberculosis strains.
Currently, the conventional methods such as light
microscopy and classic culture used to detect
Mycobacterium tuberculosis in Moroccan patients,
especially in less equipped regions are complex,
unreliable, labor-intensive, technically challenging
and time-consuming. In that context, we are actually
developing at MAScIR (Moroccan foundation for
Advanced Science, Innovation and Research) and
for the first time in Morocco a new tB detection test
based on loop-mediated isothermal amplification
technology (LAMP). tB-LAMP assay has several
advantages that make it relevant and attractive as
a diagnostic platform for resource-poor settings:
it is specific, sensitive, highly specific, fast, and it
generates a result that can be detected directly bythe
naked eye. therefore, due to its easy operation
34
without sophisticated equipment, tB-LAMP assay is
so simple to use in small-scale hospitals, primary care
facilities, and clinical laboratories in Morocco as a
point-of-care TB diagnosis test. Our test based on the
amplification of IS6110 insertion sequence, specific
to Mycobacterium tuberculosis complex (MtBC),
has been successfully tested on DNA extracted
from culture and also directly from decontaminated
sputum.
on the other hand, to counter the alarming increase
in the global incidence of MDR-tB infection, a need
for methods that can rapidly and effectively identify
drug-resistant cases becomes an emergency. thus,
we are currently developing a highly sensitive,
accurate, and rapid and an affordable assay, based
on qPCR-HRM technology, able to detect mutations
in rpob gene responsible of rifampicin resistance to
Mycobacterium tuberculosis.
OP2
EVALuATION OF FLuOROTYPE MTB FOR
ThE DETECTION OF MYCOBACTERIuM
TuBERCuLOSIS COMPLEx DNA IN PARAFFINEMBEDDED BIOPSIES
Zaira Moure1, josep Castellvi2, Adrian Sánchez3,
Maria Concepción Marina1, Maria Teresa Tortola
Fernandez1
1
Department of Microbiology
2
Department of Pathology
3
Department of infectious Diseases
Introduction and Objective: Sometimes, tuberculosis disease is not suspected and clinical samples
are not collected for culture, in these cases the only
available material is paraffin-embedded biopsies
(PEB). Recently, the Fluorotype MtB (FtMtB) (Hain
Lifescience) a real-time PCR has been introduced
for detection of Mycobacterium tuberculosis complex
(MTBC) from respiratory and non-respiratory
clinical samples. Our objective is to evaluate
FTMB in PEB.
Material and methods: A retrospective study
was performed at the vall d’Hebron university
Hospital (HuvH) from August 2012 to February
2015. Specimens were collected from patients
with histopathological results compatible with
mycobacterial infection. Mycobacterial culture was
not requested in any sample.
Clinical specimens. We studied 17 PEB from joint
(1), brain (1), endometrium (1), liver (1) bone (1),
synovium (1), pleura (1), larynx (1), intestine (2), lung
(2), and lymph node (6). DNa extraction. The PEB
were deparaffinized and DNA extraction was done
by the qIAamp DNA FFPE tissue kit ® (qIAGEN,
Hilden, Germany). PCR amplification. The DNA
obtained was amplified by the following amplification
methods: FtMtB and nested-PCR and sequencing
of the hsp65 gene (Nested-PCR). All FtMtB positive
samples were analyzed by Genotype MtBDRplus
(Hain Lifescience GmbH) and Genexpert MtB/
RIF (Cepheid, Sunnyvale, CA). the commercial
methods were performed following manufacturer’s
instructions.
Results: We studied 17 PEB from 17 patients, 14 (82%)
were Spanish citizens and 3 (18%) were immigrants.
Clinical and treatment were used as gold standard.
of the 17 patients, 9 had compatible symptoms
with tuberculous disease and received treatment, 5
(55,5%) of them had FtMtB positive. Eight patients
had no clinical and treatment, and FtMtB was
negative in 5 (62,5%). We had 7 discrepant results, 3
false positives and 4 false negatives. the 87.5% (7/8)
of the FtMtB positive samples were also positive
by Genotype MtBDRplus. All samples (8) FtMtB
positive were Genexpert negative. Nested-PCR only
detected 3 non-tuberculous mycobacteria.
Conclusions: 1/ For a routine mycobacteriology
laboratory the Fluorotype MtB has shown to be
far superior to other techniques for the detection of
M.tuberculosis complex. 2/ Genotype MtBDRplus is
useful as a genotypic susceptibility test for PEB. 3/ We
highly recommend performing mycobacteria culture
not only to confirm the viability of the microorganisms
but also to perform drug susceptibility testing.
OP3
A MuLTICENTER STuDY ON ThE NEW
VERSION OF ThE GENOTYPE MTBDRsl ASSAY
FOR ThE DETECTION OF RESISTANCE TO
FLuOROquINOLONES AND SECOND LINE
INjECTABLE DRuGS
Elisa Tagliani1, Paolo Miotto1, Andrea Maurizio
Cabibbe1, Emanuele Borroni1, juan Carlos Toro2,
Mikael Mansjö2, Sven hoffner2, Doris hillemann3,
Aksana Zalutskaya4, Alena M. Skrahina4, Daniela
Maria Cirillo1
1
emerging bacterial Pathogens unit, Division
of immunology, transplantation and infectious
Diseases, San Raffaele Scientific Institute
2
Dept of Microbiology, Public Health Agency of
sweden
3
Diagnostic Mycobacteriology, research center
borstel
4
republican research and Practical centre for
Pulmonology and tb
the limited drug susceptibility test (DSt) capacity
especially in areas with high rates of resistant forms.
We conducted a multicenter study to evaluate the
performance of the new Genotype MtBDRsl v2.0
(Hain Lifescience) assay for detection of mutations
leading to fluoroquinolones (Fqs) and second-line
injectable drugs (SLIDs) resistances.
The assay targets mutations in gyra, gyrb genes
for Fq-R, and rrs and the promoter of eis genes for
SLID-R. We evaluated the performances on 228 M.
tuberculosis complex isolates (73 Fq-R, 147 SLID-R)
and 231 smear positive specimens (57 Fq-R, 63
SLID-R) comparing results to phenotypic DSt and
sequencing.
the v2.0 assay correctly identified 83.6% of Fq-R
isolates with mutations in gyrb identified in one strain
only. overall the concordance of MtBDRsl v2.0 with
phenotypic DST and gyra qRDR sequencing was
94.7% (95% CI 91.0-97.0%) and 97.4% (95% CI
94.4-98.8%), respectively.
the test correctly identified 86.4% of SLID-R isolates,
including 27/127 (21.3%) strains carrying mutations
in eis promoter only. overall, the concordance
between MtBDRsl v2.0 and phenotypic DSt for
SLID-R was 87.7% (200/228) (95% CI 79.9-91.0%),
whereas the sensitivity and specificity were 86.4%
(95% CI 79.9-91.0%) and 90.1% (95% CI 81.794.9%) respectively. In comparison with rrs and eis
sequencing concordance was 97.8% and 98.1%,
respectively.
Among the 232 respiratory specimens tested,
MtBDRsl v2.0, showed 231 valid results. the test
correctly identified 53 (92.9%) Fq-R specimens.
overall the diagnostic accuracy for Fq-R detection
was 97.0% (95% CI 93.9-98.5%), with sensitivity and
specificity of 93.0% (95% CI 83.3-97.2%) and 98.3%
(95% CI 95.1-99.4%) respectively. the test correctly
identified 56 (88.9%) SLID-R cases. the diagnostic
accuracy for SLID-R detection in specimens was
90.9% (95% CI 86.5-94.0%), with sensitivity and
specificity of 88.9% (95% CI 78.8-94.5%) and 91.7%
(95% CI 86.5-95.0%) respectively.
Compared to v1.0, the sensitivity for Fq-R remained
unvaried, whereas MtBDRsl v2.0 showed an
improvement for the detection of xDR-tB with
specificity and sensitivity of 81.8% and 80.4% for
direct and indirect testing respectively. The new
probes included in v2.0 for detecting mutations in
eis lead to higher sensitivity for detection of KAN-R
for both direct and indirect testing (96% and 95.4%
respectively) as compared to v1.0 (66.9%). MtBDRsl
v2.0 represents an useful screening tool for rapid
detection of resistance to second-line drugs and to
guide an appropriate MDR-tB treatment.
Management of multi and extensively drug resistant
tuberculosis (MDR/xDR-tB) requires accurate
analysis of molecular DR determinants to overcome
35
OP4
TAILORED GENOTYPING TOOLS BASED
ON ALLELE-SPECIFIC PCRs TARGETING
MYCOBACTERIuM TuBERCuLOSIS STRAINSPECIFIC SNPs OBTAINED FROM WhOLE
GENOME SEquENCING DATA
Laura Pérez-Lago1, Miguel Martínez Lirola2, Iñaki
Comas3, Marta herranz1, Santiago Izco1, María
jesús Ruiz Serrano1, Paula López Roa4, juan
Carlos Momo5, Lucía Biyé Ondó5, juan Eyene5,
Emilio Bouza1, Darío García de Viedma6
1
Hospital General universitario Gregorio
Marañón
2
complejo Hospitalario torrecárdenas
3
Genomics and Health unit, Fisabio Public
Health
4
Hospital Doce de octubre
5
Plan Nacional de control de la tuberculosis y la
lepra de Guinea ecuatorial
6
servicio de Microbiología clínica y
enfermedades infecciosas, Hospital General
universitario Gregorio Marañón; inst.
investigación sanitaria Gregorio Marañón, cei
campus Moncloa, Madrid, spain
Molecular epidemiology studies in tB are generally
based on the application of the same fingerprinting tool
to all the Mycobacterium tuberculosis (MtB) isolates
in a population. In some cases, specific PCRs have
been developed to track selected strains, but this was
only possible when a specific genetic feature made it
possible to target them. Whole genome sequencing
(WGS) enables us to identify singular features (strainspecific SNPs) for any MtB strain of interest and to
develop allele-specific-oligonucleotide (ASo)-PCRs
tailored to detect relevant strains. We evaluated
the efficiency and flexibility of this new strategy in 3
different scenarios. First, we developed a multiplex
ASO-PCR to track the most actively transmitted strains
in a population directly from respiratory specimens.
The test was robust enough to be transferred to the
local laboratory addressing that transmission problem
to accelerate the availability of results. It prospectively
identified new cases infected by these strains and
retrospectively detected previously unidentified cases.
Second, we optimized a multiplex ASo-PCR targeting
4 strain-specific SNPs to quickly track in Madrid the
presence of the high-risk Beijing strain responsible
for a severe outbreak in Gran Canaria (GC). An alert
was activated after diagnosis of a tB case in Madrid
with an active infection by the GC strain resulting
from nonadherence to therapy during the previous 8
years. our test quickly tracked the GC strain, thus
enabling us to analyze, within 1 week, 868 stored
isolates from the last 6 years directly from a boiled
crude extract. Only 2 recently diagnosed cases were
infected by the strain and not related to our case, thus
challenging our assumption that the Beijing strain from
the GC outbreak was highly transmissible. Finally,
another ASo-PCR was designed to specifically
36
track a prevalent MDR strain in Equatorial Guinea
by targeting 4 strain-specific SNPs. A preliminary
study identified 2 cases infected with that MDR
strain from 26 prospective cases in which we had the
remaining Genxpert mixtures used in a pilot study.
PCRs targeting SNPs from relevant strains is a novel
strategy for monitoring of TB transmission. It is based
on low-cost, rapid, and transferable tests tailored to
the challenges of different populations.
Funding: Plan Estatal I+D+I 2013-2016, ISCIII
(12/02080,13/01207) and FEDER, IiSGM II-CooPINt-2015, Ramón y Cajal and MINECo research
grants (RyC-2012-10627, SAF2013-43521-R) (to
I.C.).
OP5
PhyResSE: WEB -TOOL FOR ANTIBIOTIC
RESISTANCE AND LINEAGE DISCRIMINATION
BASED ON WhOLE GENOME SEquENCING
DATA OF MYCOBACTERIuM TuBERCuLOSIS
COMPLEx STRAINS
Viola Schleusener1, Silke Feuerriegel2, Patrick
Beckert2, Thomas kohl2, Paolo Miotto3, Andrea
M. Cabibbe3, Daniela Maria Cirillo3, Stefan
Niemann2, kurt Fellenberg1
1
bioinformatics, research center borstel,
borstel, Germany
2
borstel, German center for infection research,
borstel, Germany
3
Diseases, San Raffaele Scientific Institute, Milan,
italy
Whole genome sequencing of clinical Mycobacterium
tuberculosis complex (MtBC) strains represents the
ultimate approach for prediction of drug resistant
tuberculosis (tB), which poses a global health threat.
WGS allows for analysis of virtually all resistance
targets and simultaneously facilitates interrogation
of further targets, such asfor phylogenetic lineage
classification. However, its general use is restrained
by the challenging data analysis and interpretation.
to overcome this data to knowledge bottleneck, we
developed the web-based “PhyResSE” tool that
enables the automated interpretation of MtBC NGS
data for determination of resistance and phylogenetic
lineage classification.
In a standardized, easy-to-perform workflow
combining tools from BWA, SAMtools and GAtk,
PhyResSE processes Fastq-files to detect genetic
variants, which are known to be involved in drug
resistance development or are specific to a particular
phylogenetic lineage. Results are based on a validated
variant list and are presented in a plain language
report. the evaluation of the pipeline, based on a
collection of 92 well-characterized MtBC strains,
confirmed excellent performance of PhyResSE.
Indeed, NGS results obtained were superior to both
classical Sanger sequencing and classical typing
methods, e.g. by detection of heterogeneous SNP
calls and improved definite phylogenetic lineage
classification.
In conclusion, PhyResSE represents the first web
tool allowing the automated knowledge-based
interpretation of MtBC WGS data for determination
of resistance and phylogenetic lineage classification.
It bridges the gap between NGS data and knowledge,
thus opening the way for a wider application of WGS
in the mycobacteriological laboratory for day-to-day
use.
rifampicin resistance and can be applied routinely
particularly in resource limited settings.
OP6
ThE VALuE OF MICROSCOPIC-OBSERVATION
DRuG SuSCEPTIBILITY (MODS) ASSAY IN
ThE DIAGNOSIS OF TuBERCuLOSIS AND
DETECTION OF MuLTIDRuG RESISTANCE
Deniz Sertel Şelale1, Meltem uzun1
1
Faculty of Medicine, Department of Medical
Microbiology, istanbul university
In control of tuberculosis, inexpensive, rapid, and
reliable methods of detecting infection by and drug
susceptibility of Mycobacterium tuberculosis complex
(MtBC) are crucial. the MoDS method has a high
sensitivity for tB and multidrug resistant-tB (MDRtB),
is an inexpensive and rapid culture method, provides
simultaneous drug susceptibility testing for MDRtB.
In this study the performance of the MoDS assay in
direct detection of MtBC and its susceptibility to drugs
was compared with conventional culture methods
(Löwenstein-jensen [Lj] culture, Mycobacterial
Growth Indicator tube 960 [MGIt] system) and
standart drug susceptibility testing (MGIt system).
of the 331 sputum samples 71 (%21.45) yielded culture
positive for MtBC by MoDS assay. Sensitivity and
specificity (% 95 confidence interval) of MoDS assay
for detection of MtBC strains were %95.95 (%91.45100.44) and 98.83 (%97.52-100.15) respectively; the
sensitivity of the assay was higher (%98.55 [%95.73101.37]) in smear positive specimens. MoDS assay
detected isoniazid, rifampicin and multi-drug resistant
isolates with %87.10 (%75.30-98.90), %85.71
(%67.98-104.04) and %92.31 (%77.82-106.79)
sensitivity and %100 (%100-100), %96.49 (%91.71101.27) and %96.55 (%91.86-101.25) specifity,
respectively. Median time for culture positivity was
similar for MGIt system (8 days) and MoDS assay
(8 days) but was significantly longer with Lj culture
(20 days) (p=0.000 for both comparisons). Median
time to the availability of the susceptibility results
was significantly shorter with MoDS assay (8 days)
compared to MGIt system (20 days) (p=0.000).
Conclusion: the findings of this study indicates that
MoDS assay is an inexpensive and rapid test with
good performance characteristics for direct diagnosis
of tuberculosis and detection of isoniazid and
37
OP7
MOLECuLAR ChARACTERISTICS OF PZA
RESISTANT STRAINS FROM TBILISI, REPuBLIC
OF GEORGIA, A hIGh BuRDEN MDR-TB
SETTING
Sarah Sengstake1, Indra Bergval1, Nino
Bablishvili2, Nestani Tukvadze2, jessica De Beer3,
Rusudan Aspindzelashvili2, Dick van Soolingen4,
Richard Anthony1
1
Kit biomedical research, royal tropical
institute, Amsterdam, the Netherlands
2
National tb reference laboratory, National
center for tb and lung Diseases, tbilisi,
Georgia
3
National tuberculosis reference laboratory,
National institute for Public Health and the
environment (rivm), bilthoven, the Netherlands
4
environment, bilthoven; Department of Medical
Microbiology, radboud university Nijmegen
Medical centre, the Netherlands
Large numbers of patients infected with a multidrug or
extensively-resistant M(x)DR-tB strain require new
treatment regimens. Almost all current first line tB
regimens including those currently being evaluated in
clinical trials include pyrazinamide (PzA). the activity
against non-replicating bacteria of PzA makes it a
valuable drug for current and future drug regimens.
However, recent studies reveal high numbers of
PzA resistant strains in MDR-tB patients. this could
compromise the effectiveness of currently developed
and tested new drug regimens that include PzA. We
have assessed PzA resistance in samples collected
at the National Tuberculosis Reference Laboratory in
tbilisi, Republic of Georgia from patients diagnosed
with pulmonary tB. the high burden of MDRTB prioritises this setting for clinical trials and/or
administration of new tB treatment regimens, but
data on PzA resistance is lacking.
We analysed 47 MDR-tB isolates for phenotypic
susceptibility to PzA and, when strains were
resistant, the pncA gene was sequenced. thirty-three
(33/47, 70.2%) MDR-tB isolates were phenotypically
resistant to PzA and all carried a mutation in pncA. In
total 17 pncA polymorphisms were identified in these
isolates of which 14 (14/17, 82.3%) were unique. the
remaining three mutations were clustered among
19 isolates of which 15 formed two MIRu-vNtR
clusters. The high heterogeneity of pncA mutations
suggests dissemination after acquisition of PzA
resistance. Deep sequencing data reveals that in
the majority of these strains the mutations in pncA
were fixed making it less likely that these mutations
38
were recently acquired in the patient. the pncA gene
of three strains showed traces of multiple genotypes
(wildtype and/or additional resistance mutations),
which could indicate de novo mutations in pncA,
acquired within the patient after initial infection with a
PzA resistant strain.
our findings support indications for both scenarios:
transmission as well as acquisition of PzA resistance.
Analysis of larger strain collections will provide
insights about the origin of PzA resistance in clinical
isolates. A better understanding of the dynamics of
PzA resistance in clinical settings may impact on
patient management and treatment policies and may
guide the administration of new drugs.
OP8
ThE ROLE OF MDR TB IN EMERGENCE OF
RETREATMENT TuBERCuLOSIS CASES
Anda Nodieva1, Inta jansone2, Ilva Pole3, Girts
Skenders3, Matiss Bauskenieks2, Ilze Morozova3,
Iveta Ozere4, Vaira Leimane3, Viesturs Baumanis2,
Renate Ranka2
1
Department of infectology and Dermatology,
Riga Stradiņš University; Bauska Hospital
2
latvian biomedical research and study centre,
university of latvia, riga, latvia
3
clinic of tuberculosis and lung Diseases, riga
east university Hospital, latvia
4
Riga Stradiņš University; Centre of Tuberculosis
and lung Diseases, riga east university
Hospital
Introduction: Multi-drug resistance (MDR) is much
higher among retreatment tuberculosis (tB) cases
than among new cases in Latvia and globally.
Aim: to find out the pathogenesis and risk factors for
recurrent TB cases.
Design and methods: Retrospective case control
study. Non-probability convenience sampling of
retreatment cases (RC) relapses, retreatments
after failure and default for cases with available
M.tuberculosis (MT) cultures for current and previous
TB episodes during 1999-2008. Drug resistance
(DR) was determined by the absolute concentration
method on Lowenstein-Jensen medium and/or
BACTEC culture. The H37Rv strain was used as a
“gold standard” for susceptible strains. Molecular
typing of Mt isolates was performed by analysis of
restriction fragment length polymorphism (RFLP) and
spoligotyping, compared in program BIoNuMERIC.
As were defined Cases (RI) - tB RC with genotipically
different Mt in both episodes, suggesting reinfection
with different strain. Controls (RA) tB RC with
genotipically identical Mt in both episodes, suggesting
reactivation.
Results: From 82 RC analyzed in 78% Mt strains of
recurrence episodes are different from initial episodes,
suggesting exogenous reinfection. Previous episode
of RI cases: drug-susceptible (DS) 72%, DR non-MDR
16%, MDR 12%, including xDR 3 cases (8% from
MDR), hospitalization 223 days in average during
1999-2002. these cases in 77% become reinfected
with MDR Mt strains, predominantly Beijing and
LAM9 genotype. Previous episode of RA controls:
DS 41%, DR non-MDR 24%, MDR 35%. Current
episode of RA controls: DS 29%, DR non-MDR 24%,
MDR 47%, including xDR 3 cases (38% from MDR),
prevalence of Beijing and LAM9 genotype. Average
hospital stays 189 days, mostly during 2003-2007.
Conclusion:
1. Most part of tB recurrences was due to the
exogenous reinfection.
2. tB patients, especially non-MDR were at risk for
recurrence due to the reinfection with MDR Mt
strains during prolonged hospitalization.
3. As a risk factor for “true” TB relapse drug resistant
Mt in the previous episode is suspected.
4. To decrease risk of TB transmission in hospitals
rapid molecular methods to detect Rifampicin
resistance, strong infection control measures and
ambulatory treatment should be enforced.
5. Mt genotyping is essential tool for molecular
epidemiology.
OP9
PYRAZINAMIDE RESISTANCE IN
MYCOBACTERIuM TuBERCuLOSIS FAILS TO
BITE?
Richard Anthony1, Alice den hertog1, Sarah
Sengstake1
1
Kit biomedical research, royal tropical
institute, Amsterdam, the Netherlands
For most widely used anti-mycobacterial drugs, a
limited number of mutations have emerged that are
responsible for the vast majority of transmitted drug
resistance in M. tuberculosis (MtB). there is one
notable exception: pyrazinamide (PzA). the list of
mutations known to confer PzA resistance in clinical
strains is long and growing, and despite the fact that
rates of 50% PzA resistance in MDR are frequently
reported, there is very little clustering of mutations.
Whereas this has received a lot of attention with
respect to the difficulty of developing practical PzA
resistance assays, the reasons behind this lack
of clustering itself has been relatively overlooked.
Despite the frequent acquisition of PzA resistance, the
responsible mutations have apparently for decades
remained incompatible with epidemic spread, and
thus appear to have a fitness cost in vivo.
This curious and poorly explained lack of clustering
and evolutionary selection of successful PzA
resistance mechanisms deserves attention. We
will present the available evidence supporting our
hypothesis that PzA resistant strains have a reduced
ability to spread and are often dead end mutations,
and present a number of testable predictions based
on this hypothesis.
OP10
ThE ROLE OF ThE BEIjING AND h4/uRAL
GENOTYPES IN ThE EPIDEMIOLOGY AND
TREATMENT OuTCOMES OF MuLTIDRuGRESISTANT TuBERCuLOSIS
Valeriu Crudu1, Elena Romancenco2, Ecaterina
Noroc3, Liliana Domente1, Sofia Alexandru1,
Dumitru Chesov1, Viorel Soltan4, Stefan
Niemann5, Christoph Lange5, M. Merker5, Richard
Garfein6, Timothy Rodwell6, Antonino Catanzaro6,
Constantin Rimis1
1
Phthisiopneumology institute
2
Phthisiopneumology institute, chisinau,
Moldova
3
Phthisiopneumology institute “chiril Draganiuc”
4
center for Health Policies and studies,
state Medical and Pharmaceutical university
“N.testemitanu”
5
borstel, Germany
6
university of california, san Diego
Background: The prevalence of multidrug-resistant
tuberculosis (MDRtB) is a serious problem, having
major public health and economic consequences
in the R.Moldova. Beijing genotype strains of
M.tuberculosis (Mtb) are associated with drug
resistance, particularly MDRtB and their prevalence
is increasing worldwide.
The aim: to provide information about the genetic
diversity and prevalent genotypes of Mtb in a high
MDRtB burden country. this study has used a
combination of information: genotyping of Mtb
strains, drug resistance surveillance data and
treatment outcomes correlated with genotype and
drug resistance pattern.
Methods: 13278 M.tuberculosis strains were
isolated from tB patients in Moldova from 2008 to
2012. Drug susceptibility assays were performed
for 90,7% (n=12038) of strains. A total of 222 Mtb
strains isolated during this period were genotyped,
using IS6110-RFLP, spoligotyping and MIRu-vNtRs.
74 of isolated were sensitive to first line drugs and
148 samples were resistant to at least isoniazid and
rifampin.
Results: Prevalence of MDRtB between New
and Re-treatment TB Cases during the period
study was on very high level: 2008-23.8% (n=266)
and 59.0%(n=629), 2009 - 22,6% (n=291)
and
69.9%(n=825),
2010-23.7%(n=349)
and
65.2%(n=770), 2011-29.2%(475) and 63.4%(n=552),
39
2012-23.7%(n=301) and 62.7%(n=603). Among the
74 sensitive strains, the genotypes identified were:
29.7% (n=22) Beijing, 17.6% (n=13) LAM, 16.2%
(n=12) H4/ural, 8.1% (n=6) Haarlem, t1&t3 6.8%
(n=5), and types x, H1, and Cameroon were 1.4%
(n=1) each. thirteen (17.6%) strains could not be
classified based on their spoligotype pattern and were
labeled as “indeterminate” (was named “Moldova like
H37Rv”). Among 148 MDR-tB strains, the genotypes
identified were: H4/ural - 50.7% (n=75), Beijing –
43.2% (n=64), LAM - 1.4% (n=2), x - 0.7% (n=1)
and “indeterminate” - 4.1% (n=6). From 148 MDRtB
strains, 71 strains were also resistant to second line
drugs (xDRtB), among those 49.0% (n=35) were
Beijing and 35% (n=25) were H4/ural genotypes.
the level of xDRtB increases considerable during
these years. In 2008 the level of xDR among MDRtB
patients was 7.4,0% and increased up to13,8% in
2011. the treatment success of M&xDRtB cases
was very low and mortality rate very high –49.0%
and 20.4% (81,7% and 6.6% in pandrug-susceptible
cohort).
Conclusion: M&xDRtB are a serious problem in the
R. Moldova, having major public health and economic
consequences. the increased number of tB resistant
cases influences of the treatment outcomes of these
patients. Beijing and H4/ural genotypes of Mtb
are predominating among MDRtB patients and
significantly are associated with xDRtB (P<0.001).
the accumulation of a greater number of MDR strains
in society can lead to the infection of population and
increase the number of xDRtB cases.
40
TB EPIDEMIOLOGY I
OP11
uRAL FAMILY OF MYCOBACTERIuM
TuBERCuLOSIS: NOT SLEEPING BEAST
ANYMORE?
Igor Mokrousov1
1
st. Petersburg Pasteur institute
Mycobacterium tuberculosis population structure
is predominantly clonal and its families may follow
different paradigms of evolution and adaptation.
Here, I systematically and critically review trends in
evolution of pathobiology and phylogeography of the
less popular and seemingly nonexciting ural family.
Genetic, phenotypic and other data on ural family
isolates were extracted from original publications
in order to trace their distribution both at global and
within-country levels. Recent reports from the ural
region in Russia confirm my previous assertion of
the smaller than initially assumed rate of the ural
genotype in this area. In contrast, its frequencies
peak in southern ukraine and Georgia, up to 19%.
Accordingly, North/East Pontic area may have been
the area of origin and primary dispersal of the ural
family in Eurasia. the MIRu-vNtR based minimum
spanning tree of ural strains appears to lack any
phylogeographic structure but this may be biased by
different size of the local collections.
ural strains are not marked with exceptional
pathogenic capacities and increased transmissibility
as demonstrated by prison based studies, mice and
macrophage models, epidemiological studies, autopsy
and human genetics studies. As a result, geographic
distribution of the ural family is still circumscribed
mainly to its area of origin and primary dispersal in
central Eurasia.
on the other hand, a non-association with multidrug
resistance appears more controversial. Indeed metaanalysis of studies across former Soviet union shows
decreased odds of being MDR for ural versus Beijing
families (P<0.00001) and similar prevalence of MDR
in ural and other non-Beijing genotypes (P=0.8).
However, MDR ural strains start to dramatically
prevail in some parts of Eastern Europe and European
Russia. It may be that a more hazardous drugresistant sublineage of the ural family commences to
emerge, as yet, at regional scale.
Acknowledgement: Russian Science Foundation
(project 14-14-00292).
OP12
MAP AFRICAN TB - POPuLATION STRuCTuRE
AND EVOLuTION OF MYCOBACTERIuM
TuBERCuLOSIS COMPLEx IN AFRICA
Patrick Beckert1, Ellen Bruske2, Florian Gehre3,
Elisabeth Sanchez-Padilla4, Véronique N. Penlap
Beng5, Abraham Alabi6, Bertrand Lell6, Thomas
A. kohl1, Francine Ntoumi7, Matthias Frank2,
Maryline Bonnet4, Bouke C. de jong3, Andrea
Rachow8, Michael hölscher8, Stefan Niemann1
1
borstel, Germany
2
institute for tropical Medicine, German center
for infection research, university of tuebingen
3
institute for tropical Medicine,
Mycobacteriology unit
4
epicentre
5
laboratory for tuberculosis research,
university of yaoundé i
6
centre de recherches Médicales de lambaréné
(cermel), Albert schweitzer Hospital
7
Fondation congolaise Pour la recherche
Médicale
8
Department of infectious Diseases & tropical
Medicine, German center for infection research,
university of Munich
According to the World Health Organization an
estimated number of 2.8 million people were newly
infected by tuberculosis (tB) and 690,000 died
from tB in Africa (2013). Even more worrisome is
the emergence of drug resistant Mycobacterium
tuberculosis complex (MtBC) isolates in Africa. Since
2011 the number of TB patients infected with multidrugresistant (MDR), MtBC strains, strains resistant to at
least the two most effective antibiotics isoniazid and
rifampicin, has doubled. Especially in Sub-Saharan
Africa with its high rate of HIv coinfected people,
the effective transmission of MDR tB is a threatening
scenario. To get an insight in transmission dynamics
of TB in Africa we analyze the population structure of
the MtBC in Africa using molecular typing methods
(spacer oligonucleotide typing (spoligotyping), 24-loci
mycobacterial interspersed repetitive units - variable
number of tandem repeats (MIRu-vNtR) typing).
These methods are ideally suited to analyze the
population structure and chains of transmission of
MtBC isolates.
So far 2,900 African MtBC isolates from 14 countries,
originating from Western Africa (420), Central Africa
(1,140), Eastern Africa (930) and Southern Africa
(450) have been genotyped. In addition to classical
genotyping the genome of selected isolates is
41
analyzed by whole-genome sequencing (WGS).
the first analysis of the population structure revealed
that 58% of MtBC isolates belong to a cluster of
strains. Clustering of strains indicates patient-topatient transmission of tB; this can be seen in all
investigated countries. In addition to that crossborder transmission of antibiotic susceptible MtBC
isolates in Kenya and Tanzania as well as Gabon
and Cameroon was observed. Furthermore, analysis
of isolates from Mozambique, Gabon and Swaziland
showed active transmission of clonal MDR strains
within the local population. At a first glance, we
had an in-depth look into isolates from Gabon and
Swaziland. We further analyzed those isolates by
WGS and could confirm effective patient-to-patient
transmission of MDR strains in two African countries.
to prevent further spreading of the MDR strains in
Gabon we established a molecular test assay to
rapidly detect the MDR strains in Gabon. therefore
we identified, based on the WGS data, a specific
single nucleotide polymorphism (SNP) for the MDR
cluster and designed a real-time-PCR based assay
to detect this SNP.
In conclusion, our study identified transmission of
different MtBC lineages all over the African continent
as well as spreading of already MDR strains in some
countries. these results give a first impression of
the population structure and transmission of MtBC
isolates in Africa. to prevent further spreading of tB,
especially MDR tB in Africa we will continue our study
and aim to establish a database of the population
structure of the MtBC in Africa.
OP13
ChALLENGES IN SETTING-uP MOLECuLAR
DIAGNOSTIC TOOLS FOR TuBERCuLOSIS
DRuG RESISTANCE DETECTION AND
EPIDEMIOLOGY STuDIES IN LOW-INCOME
COuNTRIES
Voahangy Rasolofo1, Marie Sylvianne
Rabodoarivelo1, Noël Ratovonirina1, Solohery
Razafimahatratra1, juan Carlos Palomino2,
Christophe Sola3, Fanjasoa Rakotomanana4,
Anandi Martin2
1
unité des Mycobactéries, institut Pasteur de
Madagascar, Antananarivo, Madagascar
2
laboratory of Microbiology, Department
of biochemistry and Microbiology, Ghent
university, Gent, belgium
3
institut de biologie intégrative de la cellule,
université Paris-saclay, orsay, France
4
unité D’epidémiologie, institut Pasteur de
tuberculosis (tB) is a serious health problem in
developing countries worsened by HIv co-infection
and the emergence of multidrug-resistant (MDR)
and extensively drug-resistant (xDR) Mycobacterium
tuberculosis strains. Novel molecular technologies
42
are now available for rapid screening of TB drug
resistance. Genotyping is also currently used for
molecular epidemiology studies. They are powerful
tools for the surveillance of MDR-tB, for tracking
strain transmission in a population or identifying
hotspots of TB transmission. However current
molecular tests require DNA extraction from either M.
tuberculosis isolates or ideally from sputum samples.
Moreover, in low-income countries, performing this
type of tests is often restricted by constraints of both
sputum storage and safely transport from peripheral
health centres to central laboratories. To help
overcome such problems, we recently implemented
sputum collection, transport, and DNA extraction from
different supports such as filter cards and direct smear
to study MDR-tB in low-income settings by using the
Genotyping MtBDRplus test. We also carried out a
molecular epidemiology study of TB in Antananarivo.
the objective was to assess whether it was feasible
to identify hotspots of TB transmission by combining
two methods: the determination of genotypic clusters
by M. tuberculosis strain spoligotyping with DNA
extracted from stained sputum smears, and the
identification of tB case clusters by Geographical
Information System. Because of its simplicity and
ease of transport, commercialized storage and
transport supports are different systems that have the
potential to be used widely in remote endemic areas
where specimen collection is most difficult to perform
and can potentially be used for epidemiology study.
The main goal of this presentation is to discuss the
potential use of DNA extracted from different supports
for subsequent molecular testing. We will discuss
advantages and disadvantages of these systems.
TB EPIDEMIOLOGY II
OP14
INSIGhTS FROM MOLECuLAR TYPING INTO
ThE RAPIDLY EVOLVING EPIDEMIOLOGICAL
LANDSCAPE OF TuBERCuLOSIS IN SICILY
Celestino Bonura1, Michel Gomgnimbou2,
Guislaine Refrégier3, Christophe Sola4, Caterina
Mammina1
1
Department of sciences for Health Promotion
and Mother-child care « G. D’alessandro »,
university of Palermo, italy
2
centre Muraz, bobo-Dioulasso, burkina Faso
3
orsay, France
4
institut de Génétique et Microbiologie,
université Paris-sud-cnrs, orsay, France
Epidemiology of tuberculosis (tB) in Sicily is evolving
with the most striking feature being the rapidly
increasing proportion of cases in the foreign-born
population.
An observational study of the tB notifications in the
years 2010-2014 has been performed by analyzing the
Sistema informativo delle Malattie infettive database.
A molecular epidemiological analysis of 151 isolates
of Mycobacterium tuberculosis complex (MtBC),
of which 81 from foreign-born patients, identified in
Palermo in the years 2012-2013, was also carried
out by spoligotyping and 24-loci MIRu-vNtR typing.
Susceptibility testing to 1st line antiTB drugs was also
carried out.
In the period unders tudy, 1124 new tB cases have
been notified in Sicily with a mean annual notification
rate of 4.50 cases per 100.000 inhabitants (from
3.96 in 2011 to 5.00 in 2014). the proportion of tB
cases in the foreign-born individuals was increasing
from 36.4 in 2010 to 58.8 in 2014. The median age
of foreign-born patients was significantly lower than
the Italian-born (30.75 vs. 48.92 years, P < 0.001).
the distribution by age class was also significantly
different, with the largest proportion of tB cases
among the foreign-born sub-population in the 1544 age class, whereas the Italian-born cases were
equally distributed in the 15-44, 45-64 and >64 age
classes. The diagnostic delay proved to be longer in
Italian-born than in foreign-born patients (104.15 vs.
89.95 days).
Fourteen lineages and 33 sublineages showing a
different distribution among the two patients subpopulations were detected. only two MDR isolates
were identified from an Italian born elderly patient and
an Eritrean young patient (Beijing lineage).
TB epidemiology in Sicily is a complex mix of
reactivation in the autochthonous population and in
sub-populations of immigrants from high endemic
countries and recent transmission among vulnerable
subgroups of both foreign and Italian origin. An
integrated approach using both conventional and
molecular tools is necessary to accurately assess and
monitor TB epidemiology in this geographical area.
OP15
COMPLEx GENOMIC DIVERSITY OF
MYCOBACTERIuM TuBERCuLOSIS IN EThIOPIA
Iñaki Comas1, Sebastien Gagneux2, David
Stucki3, Douglas Young4, Abraham Aseffa5,
Stefan Berg6
1
center for Public Health research (csispFisabio), ciber epidemiology and Public Health
2
Department of Medical Parasitology and
infection biology, swiss tropical and Public
Health institute, university of basel, switzerland
3
swiss tph
4
Mrc National institute for Medical research, Mill
Hill, london
5
Amauer Hansen research insitute
6
Animal and Plant Health Agency
Recent publications have highlighted the diversity of
the tuberculosis bacillus in Ethiopia. However a deep
genomic sampling of this diversity has not been done
before. We reasoned that by rationally choosing a
collection of strains representing common and rare
strain types in Ethiopia we could at the same time
describe the genomic diversity of the bacteria in the
region and help to distinguish between competing
models of the timing and origin of the MtBC. As
expected analyses corroborates the highly complex
population structure of the pathogen paralleling
that observed in human populations. This complex
population structure paradoxically adds more
uncertainties about when and how tuberculosis
became a global disease.
Funding: Ramón y Cajal and MINECo spanish
research grants (RyC-2012-10627, SAF201343521-R), Medical Research Council uk (grant
u.1175.02.002.00015.01), the Swiss National Science
Foundation (PP0033-119205), the National Institutes
of Health (AI090928 and HHSN266200700022C),
Wellcome trust, uk (grant number 075833/A/04/z).
43
OP16
COMPARATIVE INTRADERMAL TuBERCuLIN
TEST IN DAIRY CATTLE IN ThE NORTh OF
ECuADOR AND RISk FACTORS ASSOCIATED
WITh BOVINE TuBERCuLOSIS IN ANIMAL AND
huMANS
Freddy Proano-Perez1, Lenin Ron-Garrido1,
Ricardo Benitez-Capistros1, Francoise Portaels2,
Leen Rigouts2
1
universidad De las Fuerzas Armadas espe;
universidad central Del ecuador
2
institute of tropical Medicine of Antwerp
Bovine tuberculosis (BtB) is a chronic zoonotic
disease caused by Mycobacterium bovis, which has a
worldwide distribution in domestic and wildlife animals.
The aim of this study was to evaluate the state of the
disease in cattle and humans, and evaluate the risk
factors associated in the transmission of BTB. 20 dairy
herds comprising 2,022 cattle were selected from
medium and large size herds located in Mejia canton,
the major dairy production area in northern Ecuador.
Each animal was tested using the comparative
intradermal tuberculin test (CItt); and one year later,
a follow up test was performed in the same herds.
In humans, the study was carried out in 157 people,
conformed by farm workers and slaughterhouse
workers, with the purpose to evaluate the prevalence
of Mycobacterium spp. in risk populations by
tuberculin skin test (tSt) and analyse risk factors.
the true annual incidence was 1.70%, and the true
prevalence was 7.41% and 7.13%, respectively. the
prevalence was 0.27% and 0.57% in medium sized
herds, respectively, compared with 8.63% and 8.43%
in large herds. the number of skin test–positive
cases also increased significantly with age, contacts
with other species of animals, and introduction of new
cattle. Herd prevalence was 55% in the first year and
65% in the follow up study. Mycobacterium spp. was
estimated in 29% of the farm and slaughterhouse
workers. the results establish a significant association
among the positivity of TST to masculine gender
(P=0,026), consumption of milk coming from farms,
consuming raw milk, and consumption of elaborated
cheeses made by hand. Consumption of raw milk
gave an or=4,65. this study shows the necessity
for a national BTB control program in Ecuador which
should be supported by sanitary policies to allow
the identification of the endemic areas nationwide.
In addition, due to the lack of knowledge in cattle
farmers about this zoonoses, education of farm and
abattoir workers about the disease could help prevent
new cases in animals and humans, and help improve
the use of preventive measures.
44
OP17
WhAT hAVE STuDIES ON ThE PROTEIN
ExPRESSION OF MYCOBACTERIuM
TuBERCuLOSIS BROuGhT uS?
jeroen de keijzer1, P. de haas2, A. Mulder2, j.
de Beer2, A. de Ru1, P. van Veelen1, Dick van
Soolingen3
1
leiden university Medical centre
2
environment
3
environment, bilthoven; Department of Medical
Microbiology, radboud university Nijmegen, the
Netherlands
the field of mass spectrometry (MS)-based proteomics
focusses on the identification and quantitation of
proteins without the need of antibodies, which are often
lacking for M. tuberculosis proteins. The astonishing
development in MS technology greatly enhanced the
potential of proteomics in life sciences.
In our laboratory, we use the so called “bottom-up”
proteomics workflow, where proteins are digested and
the resulting peptides are sequenced. We have applied
this principle for the classification of mycobacteria by
species-specific peptide sequences and to confirm or
correct existing genome annotations.
With the usage of mass-tags we can quantitatively
study the proteome of M. tuberculosis. We have
applied a quantitative proteomics approach to study
the M. tuberculosis Beijing family, which consists out
of the more ancient (atypical) and modern (typical)
sublineage. Especially the more recently evolved
modern Beijing strain has been associated with
(multi)drug resistance and the ability to cause and
spread tuberculosis. In this study, we systematically
compared protein expression levels of ancient and
modern Beijing strains. Despite the uniform protein
abundance ratios in both sublineages, we identified
two proteins as differentially regulated, which could
explain the apparent increased adaptation of modern
Beijing strains. In addition, we demonstrate the
preexistence of several, possibly unique, drug efflux
pumps in the proteome of M. tuberculosis Beijing.
To further examine how protein regulation may help
M. tuberculosis Beijing strains cope with antibiotics,
we examined the initial response of M. tuberculosis
Beijing B0/W148 to rifampicin. the induction of the
DosR regulon and regulation of post-translational
modifications demonstrated that phenotypic drug
tolerance is likely to play a role in the success of this
particular Beijing strain. In fact, for the first time, we
provide evidence that conversion of asparagine to
aspartic acid in the rifampicin resistance determining
region, by a chemical process known as deamidation,
might be a common cause of rifampicin tolerance.
As rifampicin treatment is capable of inducing a
dormant phenotype in vitro it will be of the essence to
develop drugs that act bactericidal in the case of latent
tuberculosis. Thioridazine is known to be effective
against the dormant form of M. tuberculosis. Moreover,
thioridazine displayed strong synergistic effects with
other drugs. It had been hypothesized that this might
be caused by a inhibition of antibiotic extruding efflux
pumps. However, unbiased proteomic analysis of
thioridazine treated M. tuberculosis showed that a
change in cell envelope permeability is responsible
for a rapid influx of chemical compounds.
MS-based proteomics opened the way to study
mycobacterial
protein
regulation.
therefore,
we strongly believe that proteomics will play an
increasingly large role in future studies.
OP18
IDENTIFICATION OF MuTATIONS AFFECTING
CANDIDATE GENES POTENTIALLY INVOLVED
IN PhENOTYPIC RESISTANCE TO BEDAquILINE
AND DELAMANID IN M. TuBERCuLOSIS
Simone Battaglia1, Andrea Maurizio Cabibbe1,
Elisa Schena1, Emanuele Borroni1, Paolo Miotto1,
Daniela Cirillo1
1
The upsurge of multi- and extensively- drug resistant
tuberculosis (M/xDR-tB) is reducing dramatically the
spectrum of available drugs for treatment. Therefore
effective compounds are needed. Among new anti
tB drugs, bedaquiline (BDq) and delamanid (DLM),
are conditionally approved. Mutations responsible
for BDq-R are found in atpE gene. However, other
spontaneous BDq-R mutants harbour mutations in the
non-target Rv0678 gene, a negative regulator of the
MmpS5-MmpL5 efflux pumps. Foremost mutations
in Rv0678 are related to clofazimine resistance.
DLM is a pro-drug requiring intracellular activation
by Rv3547 protein. Mutations in Rv3547 gene or in
fbia, fbib, fbiC and fgd1 genes (F420 biochemical
pathway) are involved in DLM-R phenotype. taking
advantage of Whole Genome Sequencing (WGS)
approach, we analysed eight genes putatively
involved in BDq-R and DLM-R on 219 MtB isolates
(79 susceptible to rifampicin (RIF) and isoniazid
(INH), 11 RIF-R/INH-S, 96 MDR, 11 MDR+AG-R, 12
MDR+Fq-R and 10 xDR) representing 13 different
lineages. We focused our attention on atpE, Rv0678,
45
mmpL5, mmpS5 genes for BDq-R; ddn, fgd1, fbia,
fbib and fbiC genes for DLM-R. our data allowed
to identify several new non-synonymous mutations
in genes putatively involved in BDq-R. In particular,
although no mutations were observed in atpE, the
other genes analysed carried substitutions: 7 strains
have mutations in Rv0678 gene (v3I, N4t (2), M17v,
L32S, G87R, R134G); 18 in mmpL5 (P70L, D128N
(3), S325G, A349t, E552G, L634q, F696L (8),
q702k, q707k); 2 in mmpS5 (G109S). We observed
mutations possibly involved in DLM-R in: Rv3547 (3
isolates: R72W, E83D, W88StoP); fgd1 (1: G314E);
fbia [8: q120R (4), t302M (4)]; fbib [62: F220L (56),
D315A (1), k448R (6)]; fbiC (3: v41M, t273A, I693v).
From this subset of data, we did not observe particular
correlation among mutations in candidate genes for
BDq-R or DLM-R and the resistance pattern and/
or genotype of the strains included in the study.
Previously reported lineage-specific substitution in
fgd1 (k270M: Haarlem; k296E: West Africanum 2)
and mmpL5 (D767N: Beijing) were also detected.
Phenotypic testing is under investigation to better
understand the potential role of these new mutations
in the emergence of BDq-R and DLM-R. the WGS
approach showed its usefulness in analysing a wide
range of DR-related genes in a short time, allowing to
perform a fast analysis of mutations linked to anti-TB
drug-resistance.
OP19
DETECTION OF PYRAZINAMIDE
hETERORESISTANCE IN M. TuBERCuLOSIS
jim Werngren1, Mikaela Glader2, Sven hoffner3
1
unit for Highly Pathogenic bacteria, Department
of Microbiology, Public Health Agency of sweden
2
Dept. of Medical biochemistry and Microbiology,
uppsala university
3
Dept. of Microbiology, tumour and cell biology,
Karolinska institute
Aim: Pyrazinamide (PzA) is a first line key agent
in the effective treatment of tB, including PzA
susceptible MDR-tB. occasionally, tB patients might
have mixed infections with both drug sensitive and
resistant strains. This is termed heteroresistance. If
10% of the bacterial population is resistant to PzA,
there is an increased risk for poor treatment outcome.
The aim of this study was to evaluate the ability of
the three established drug susceptibility testing
(DSt) techniques BACtEC MGIt 960, Wayne’s
pyrazinamidase test and Sanger sequencing of the
pnca gene to detect 10% PzA heteroresistance.
Methods: Mixed cultures of the fully drug susceptible
M. tubertculosis H37Rv reference strain and two
laboratory generated isogenic H37Rv mutants (with
C475G and T254C pnca mutations, respectively) were
made in proportions of 100%, 10%, 5% and 1% of the
PzA-resistant (PzA-R) strain. Corresponding mixed
cultures were also made using one drug susceptible
46
and one PzA-resistant MDR clinical isolate with the
T62G pnca mutation, both belonging to one specific
MIRu cluster. Additional mixes of 50%, 75%, 90%
and 99% of the PzA-R strains were prepared for the
Wayne’s test. tests were for all methods performed in
duplicates at two separate occasions.
Results: using the MGIt system, the in vitrogenerated PzA-R strains were generally detected at
a 5% proportion while the clinical PzA-R isolate only
was detected at the critical 10% proportion, except for
one test occasion. Sanger sequencing was unable to
detect 10% PzA heteroresistance. Wayne’s test also
failed to detect the critical level of 10% PzA resistance,
instead it displayed misguiding results determining
highly resistant samples as susceptible.
Conclusion: Heteroresistance is caused by present
drug resistance development and/or dual infections
with one resistant and one susceptible strain. Mixed
infections with resistant strains may occur in up to
20% of all tB cases in high burden areas, according
to limited data. Our study showed that only the
phenotypic BACtEC MGIt system was capable
in determining the critical proportion of 10% PzA
resistance, whereas, neither the Sanger nor the
Wayne’s test were successful in this respect. this
indicates a need for diagnostic tools with increased
sensitivity to determine heteroresistance in M.
tuberculosis.
keywords: Pyrazinamide, heteroresistance, tB, drug
resistance, DSt
hOST – PAThOGEN ITERACTION
OP20
ThE IMPACT OF LIPIDS IN ThE GLOBAL
GENETIC ExPRESSION OF MYCOBACTERIuM
TuBERCuLOSIS DuRING ITS ADAPTATION TO
DORMANCY
Diana Angelica Aguilar Ayala1, Dieter
De Coninck2, Dieter Deforce2, Filip Van
Nieuwerburgh2, juan Carlos Palomino1, Peter
Vandamme1, jorge González Y Merchand3,
Anandi Martin1
1
university, Gent
2
laboratory of Pharmaceutical biotechnology,
Faculty of Pharmaceutical sciences, Ghent
university, Gent
3
Departamento de Microbiología, escuela
Nacional de ciencias biológicas, instituto
Politécnico Nacional
It is estimated that one third of the world’s population
has latent tuberculosis infection (LtBI). LtBI is
asymptomatic; the tB bacillus becomes refractory
to antibiotics and, in low- and moderate-endemic
countries, most active tB cases arise by reactivation
of LTBI. Although several in vitro models exist to mimic
the environment of dormant bacilli during latency,
cultures grown in the presence of lipids, the most
abundant molecules surrounding bacilli in vivo,
have not been extensively explored. We considered
previous studies on gradual depletion of oxygen and
the use of long-chain fatty acids, as carbon source,
which make the bacilli switch-on their program related
to a dormant stage: slowed growth, accumulation
of lipid bodies, and development of drug tolerance.
Also the fact that Mycobacterium tuberculosis
(Mtb) accesses fatty acids and cholesterol from the
macrophage host cell and use them to establish the
infection and to survive during dormancy. Hence, the
objective of this study was to determine and analyze
the global genetic expression of Mtb in an in vitro
model of dormancy based on lipids.
Mtb H37Rv was adapted to grow in Dubos medium
supplemented with a mixture of cholesterol plus longchain fatty acids (palmitic, stearic and oleic acids) in
aerobic conditions, which was lately used as control
condition. Subsequently, an in vitro dormancy model
for Mtb, based on oxygen depletion in the presence of
a mixture of these lipids was standardized. Afterwards,
genes related to cellular cycle, immunodominant
antigens and lipid metabolism of Mtb were assessed
by qRt-PCR. to evaluate its relevant expression,
hypoxic condition’s results were compared with
those from aerobic cultures of Mtb. Finally, the global
transcriptome of Mtb during its in vitro adaptation to
dormancy in the presence of lipids was analyzed by
RNAseq. We previously reported that Mtb was able to
persist during its in vitro adaptation to dormancy in the
presence of lipids. Initial results of genetic expression
showed that during this in vitro latency model of Mtb,
the presence of lipids favored overexpression of some
genes related to virulence and its lipid metabolism
increased, leading to synthesis of triglycerides and
structural lipids of the cell wall. The transcriptome of
Mtb gene expression during its active growth and in
vitro dormancy in the lipid-rich environment is under
bioinformatics analysis and will be discussed during
the presentation.
OP21
PRELIMINARY IN SILICO-BASED INSIGhTS ON
ThE FuNCTIONALITY OF CRISPR-Cas IN M.
TuBERCuLOSIS COMPLEx
Guislaine Refrégier1, Matthieu Petrou1,
Christophe Sola1
1
institute for integrative biology of the cell,
université Paris-sud
CRISPR locus has been used extensively for more
than 20 years to provide spoligotyping profiles of
Mycobacterium tuberculosis complex isolates. The
corresponding technique is still widely used for
molecular epidemiological surveillance and clinical
microbiology, and CRISPR locus is considered to
evolve neutrally.
While their functional role was hypothesized based
on sequence similarity of spacers to phages in 2005,
it is only in 2007 that the functional role of CRISPRs
in association with the adjacent cas genes was
demonstrated in Streptococcus thermophilus: they can
provide acquired resistance against bacteriophages
by 3 steps, 1) spacer acquisition, 2) CRISPR-RNA
processing and 3) association to and degradation
of the targets. other roles have been identified in
other species: prevention of plasmid conjugation in
Staphylococcus epidermidis, expression modulation in
E. coli, Salmonella and in Pseudomonas aeruginosa.
CRISPR-Cas systems, through cas9 also serve as
genome engineering tools. However, there is still poor
information on the potential roles, if any, of CRISPRCas system in M. tuberculosis.
We first explored the potential functionality of Cas
proteins by analyzing their sequence and structure
conservation as compared to Staphylococcus
epidermidis, a species that carries the same type
of CRISPR-Cas system (type III-A). In contrast with
the well-documented absence of spacer acquisition
47
in M. tuberculosis complex, the structure of proteins
classically involved in spacer acquisition (step1,
proteins Cas 1 and Cas2) was largely conserved. the
protein involved in crRNA processing (step2, Cas 6)
was slightly shorter in M. tuberculosis but still exhibited
high structure similarity to that of S. epidermidis. The
quality of structure prediction of proteins involved in
association and degradation (step 3) was too low to
allow any prediction.
Second, mining available genomic data within
PolytB, we studied the sequence diversity inside M.
tuberculosis complex. We detected some mutations,
however with little or no impact on protein structure.
Altogether, cas genes do not seem to evolve as
pseudo-genes in M. tuberculosis complex. If they
still have a function, the well-documented absence of
spacer acquisition suggests that its function could be
different from that of other CRISPR-Cas systems.
OP22
CxCL9, CxCL10, AND IL6 CYTOkINES AS
POTENTIAL BIOMARkERS OF TREATMENT
RESPONSE IN PuLMONARY TB PATIENTS
Vladyslav Nikolayevskyy1, Yanina Balabanova1,
Irina kontsevaya2, Olga Ignatyeva2, Edita
Pimkina3, Girts Skenders4, Francis Drobniewski1
1
imperial college london
2
samara tb service, samara, russian Federation
3
infectious Diseases and tb Hospital, Vilnius,
lithuania
4
Background: tuberculosis (tB) poses a major health
problem globally causing over 9 million new cases
and 1.5 million deaths annually. To date only 2 months
culture conversion has proven effective in predicting
favourable outcomes of drug sensitive tB; no reliable
biomarkers for drug resistant and MDR/xDR tB have
been identified so far. Novel biomarkers research
is urgently needed to improve our understanding of
TB disease development and progression leading
to cure, failure or relapse. Levels of circulating proinflammatory cytokines and other molecules involved
in cellular immune response pathways as well as
their kinetics during TB infection were shown to be
different in fast and slow responders indicating their
potential role as biomarkers of treatment response
and cure.
Materials and methods: Within the Eu FP7
funded PANNEt project large longitudinal cohorts
comprising a total of 308 pulmonary drug-sensitive
and drug-resistant TB patients have been established
and followed up for up to 24 months at three sites
in Russia, Lithuania, and Latvia. In our study we
analysed levels of thirty soluble pro-inflammatory
cytokines, chemokines and growth factors in plasma
samples taken from 61 fully sensitive patients at
diagnosis and different time points during anti-TB
48
treatment using Luminex® technology. Conventional
laboratory data (smear microscopy and bacteriology)
as well as baseline clinical and epidemiologic data
was collected. Patients were classified into two
groups (fast and slow responders) based on culture
conversion at 2 months of treatment. 24 loci vNtR
genotyping was used to identify cases of reinfections
in TB patients.
Results: Levels of CxCL9 and IL-6 were significantly
higher in slow responders than those in fast responders
(p=0.0132 and p=0.0411 respectively) while CxCL10
level was significantly higher in fast responders
(p=0.0047) suggesting that CxCL9, CxCL10 and IL-6
can be considered biomarkers of treatment response.
Differences in levels of other cytokines/chemokines
between two groups were not statistically significant.
Analysis of kinetics of cytokines allowed to identify
the certain common trends, including levels of
many circulating cytokines going down during the
treatment with concentrations in slow responders
remaining higher at months 2-6 compared to those
in fast responders. Reinfection with a new TB strain
not matching the original 24 loci vNtR profile was
detected in one patient only.
OP23
spans approx. 90 kb, including the MAP specific large
sequence polymorphisms LSP14 and 15, harboring
64 genes of which 45 responded to zinc starvation.
of these, 35 were predicted to be regulated by the
major zinc metalloregulator FurB. this clustering of
zinc responsive genes in MAP is unique as it was
not found in other mycobacteria. the znGi harbors a
MAP specific operon, which showed no homologies
to genes in other mycobacteria and was predicted
to encode for two ABC-transporters (mptABC and
mptDEF) involved in metal transport. We found that
the expression of mptABC was highly induced at low
zinc concentrations. In addition, heterologous reporter
gene assays with the lacz gene under control of the
mptABC promoter in M. smegmatis (MSMEG) and in
a MSMEG∆furB deletion mutant revealed that the zinc
dependent expression was mediated by FurB via a
conserved mycobacterial FurB recognition site. thus,
this FurB regulated zinc responsive operon seems to
represent an additional zinc transporter in MAP.
*
Corresponding author. Mailing address: Institute for
Microbiology, Department of Infectious Diseases,
university of veterinary Medicine Hannover,
Bischofsholer Damm 15, 30173 Hannover, Germany,
Phone: ++49-511 8567730, Fax: ++49-511 8567697,
Email: elke.eckelt @tiho-hannover.de
EVIDENCE FOR MYCOBACTERIuM AVIuM
SSP. PARATuBERCuLOSIS SPECIFIC ZINC
TRANSPORT SYSTEMS
Elke Eckelt1, Ralph Goethe1, jochen Meens1,
Michael jarek2
1
institute for Microbiology, university of
Veterinary Medicine school Hannover
2
Genome Analytics, Helmholtz centre for
infection research
Iron and zinc are the most abundant transition metals
in all living cells and required for a multiplicity of
biological processes. Hence, availability of these
molecules is essential for survival. Host cells have
made use of this circumstance and developed a
major defence mechanism, the nutritional immunity.
An active alteration of metal concentration leads to
either intoxication or starvation of invading bacteria.
thus, maintenance of metal homeostasis is crucial in
bacterial pathogenicity and many pathogenic bacteria
have evolved sensitive regulated metal transport
systems to overcome this particular host defence
mechanism.
The ruminant pathogen Mycobacterium avium
ssp. paratuberculosis (MAP) displays a unique gut
tropism and causes paratuberculosis, a chronic
progressive intestinal inflammation. In addition MAP
displays defects in genes for mycobactin synthesis.
Hence, we were interested to find particularities
in MAP metal homeostasis. By metal starvation
experiments, subsequent RNA sequencing and
transcriptome analysis, we identified a MAP specific
zinc responsive genomic island (znGi). the znGi
49
TOPIC ENVIRONMENTAL MYCOBACTERIA
OP24
Ms1, A NOVEL sRNA INTERACTING WITh ThE
RNAP CORE IN MYCOBACTERIA
Libor krásný1, jarmila hnilicová1, Michaela
Šiková1, Martina janoušková1, Jiří Pospíšil1
1
institute of Microbiology, Academy of sciences
of the czech republic
Small RNAs (sRNAs) are molecules essential for a
number of regulatory processes in the bacterial cell.
We have characterized Ms1, a sRNA that is highly
expressed in Mycobacterium smegmatis during
stationary phase of growth. We show that Ms1
interacts with the RNA polymerase (RNAP) core that
is free of the primary sigma factor (σA) or any other
σ factor. this contrasts with the situation in most
other species where it is 6S RNA that interacts with
RNAP and this interaction requires the presence of
the primary sigma factor. We demonstrate that Ms1
functions by stabilizing the RNAP core, protecting it
against degradation. thus, Ms1 represents a novel
type of small RNAs interacting with RNAP.
OP25
INCIDENCE AND DIVERSITY OF
NONTuBERCuLOuS MYCOBACTERIA IN
MOSCOW REGION
Maria Alvarez Figueroa1, Valentina Barilo2,
Anastasiya Prokopenko3
1
central research institute for epidemiology of
Federal service for surveillance on consumers’
rights Protection and Human Well-being,
Moscow; the Moscow research and clinical
center for tuberculosis control of the Moscow
Government Health Department; Federal state
Budgetary Institution “scientific Center for
expertise of Medical Application Products”
Ministry of Health of the russian Federation
2
the Moscow research and clinical center for
tuberculosis control of the Moscow Government
Health Department
3
Moscow, russia
the incidence of tuberculosis (tB) in Russia is very
high. Reported incidence rates of TB in Russia are
68.1 in 2012, 63 in 2013 and 59.5 in 2014 as well as in
Moscow amount 40.4 in 2012, 32 in 2013 and 28.1 in
2014. Among them over 40% were microbiologically
50
confirmed. there is no statistical data of
mycobacteriosis incidence in Russia. The information
of diversity of nontuberculous mycobacteria is very
scares excepting of North-West of Russia.
The aim of this study was to determine the population
structure of clinical isolates of mycobacteria and
to estimate diversity of NtMB in Moscow region in
2012-2014.
Design/Methods: the species identification of
clinical isolates of mycobacteria had received from
inpatients of Clinic №2 of the Moscow Research and
Clinical Center for Tuberculosis control was carried
out by immunochromatographic assay (Standard
Diagnostics, korea), routine biochemical tests,
PCR-Rt (AmpliSens, Russia) and sequencing (16S
rDNA, ItS, hsp 65). All laboratories’ techniques were
identical throughout the period of 3 years.
Results: A total of 526 mycobacterial isolates, 17
(3.23%) were determined as NtMB in 2012, of 562
isolates – 30 (5.34%) were NtMB in 2013, and of
558 – 38 (6.81%) were NtMB in 2014 (p<0.01, t-test,
2012 / 2014). We found out a 1.76-fold increase in
number of NtMB in 2013 compared to 2012 and a
1.27-fold in 2014 compared to 2013.
We did not found advantage of isolation of NtMB
from patients with HIv-status. the proportion was
54.94% (9 of 17) in 2012, 40% (12 of 30) in 2013, and
47.37% (18 of 38) in HIv+ patients. there was weak
correlation between HIv- status of patients and the
diagnosis of mycobacteriosis (according an official
AtS/IDSA statement, 2006): r=+0.51.
For the three-year period were isolated a total of 85
clinical isolates from individual patients. the frequency
of M. avium complex, as expected, was higher,
67.06% (n=57). M. fortuitum have been represented
in 17.65% (n=15) and M. kansasii in 8.24% (n=7)
cases. The remaining species were isolated in single
cases: M. abscessus, M. celatum, M. chelonae, M.
gordonae, M. szulgai (each of them in 1.18%). For
the first time we managed to isolate M. colombiense
from sputum in a patient (HIv-negative) who had at
the same time cutaneous TB.
Conclusions: The use of molecular typing methods
allowed us to estimate the increasing proportion
of NtMB among clinical isolates of mycobacteria
during observed period. the most frequent species
in Moscow are M. avium complex, M. fortuitum and
M. kansasii.
OP26
FIRST GENOTYPING STuDY OF CLINICAL
AND ENVIRONMENTAL MYCOBACTERIuM
uLCERANS ISOLATES FROM FRENCh GuIANA
REVEALS uNPRECEDENTED GENETIC
DIVERSITY
Yann Reynaud1, julie Millet1, David Couvin1,
Aaron Morris2, Rodolphe Gozlan2, jean-François
Guéguan2, Nalin Rastogi1, Pierre Couppié 3, Eric
Legrand4
1
institut Pasteur, Pointe À Pitre
2
ird, Montpellier, France and cayenne
3
centre Hospitalier André rosemon, cayenne
4
institut Pasteur, Paris
Buruli ulcer is an emerging and neglected tropical
disease caused by Mycobacterium ulcerans. Few
cases have been reported so far in the Americas.
With 250 cases reported since 1969, French Guiana
is the only Buruli ulcer endemic area in the continent.
thus far, no genetic diversity studies of strains of M.
ulcerans from French Guiana have been reported.
Our goal in the present study was to examine the
genetic diversity of M. ulcerans strains in this region
by sequencing 6 previously-described variable
Number of tandem Repeat (vNtR) markers. A
total of 23 DNA samples were purified from ulcer
biopsies or derived from pure cultures, and 10
DNA samples from the environment (water, soil,
algae, aquatic plant and biofilm). A total of 4 allelic
combinations were characterized in our study
for clinical samples: genotype I which has been
described previously, genotype III and Iv which are
very similar to genotype I, and genotype II which has
distinctly different characteristics in comparison with
the other two genotypes. A total of 5 more genotypes
were described in environmental samples. This high
degree of genetic diversity appears to be uncommon
for M. ulcerans. Further research based on complete
genome sequencing of strains belonging to genotypes
I and II is in progress and should lead soon to a better
understanding of genetic specificities of M. ulcerans
strains from French Guiana.
OP27
ANALYSIS OF MYCOBACTERIuM ABSCESSuS
GENETIC VARIABILITY PROVIDED BY 14-LOCuS
VARIABLE-NuMBER TANDEM-REPEAT IN
PATIENTS WITh CYSTIC FIBROSIS
Alberto Trovato1, Daniela Maria Cirillo1, Rossella
Baldan3, Enrico Tortoli1, Stefan Niemann4,
Giovanni Taccetti5, Silvia Campana5, Lisa
Cariani6, Paola Maria Rancoita7, Thomas kohl4,
Tullia Simonetti8
1
Irccs San Raffaele Scientific Institute
borstel, Germany
5
ospedale Pediatrico Meyer
6
ospedale Maggiore Policlinico
7
università Vita salute san raffaele
8
Azienda ospedaliero-universitaria careggi
3
4
In the last decades nontuberculous mycobacteria
have been increasingly reported in cystic fibrosis (CF)
patients. Mycobacterium abscessus (MA) is the more
frequently isolated from the lung of CF patients.
Whole genome sequencing has produced evidence
supporting the splitting of MA in three subspecies: MA
abscessus, MA bolletii and MA massiliense and, very
recently, a large study has produced the first evidence
of a possible person-to-person transmission.
Our study aimed at producing a preliminary assessment
of the epidemiological value of a customized set of 14
variable-Number tandem-Repeat (vNtR) markers
selected among the most variable reported MA-vNtR
loci, in order to obtain the highest combined HGDI
(Hunter-Gaston Discriminatory Index).
A total of 122 strains, collected from 29 CF patients
attending three different Italian CF centers, were
characterized at subspecies level by sequencing the
rpoB gene and then subdivided by using 14 different
vNtR loci (MAB-1, -4, -7, -9, -11, -14, -18, -21, -23,
-24, -28, -29, tR-109 and -155).
MAB-28, initially considered for the high reported
heterogeneity, was excluded because of the high
average molecular weight of its amplicons and the
presence of incomplete repeats, which made difficult
to estimate the size on a 3% agarose gel. A good
reproducibility was obtained for all the rest of vNtR
markers. MAB-11 showed the highest allelic diversity
(0.78) followed by MAB-24 (0.75).
Crucially, this set of 13-loci perfectly matched the
subspecies classification obtained by sequencing the
rpoB gene, showing a minimum 5-locus difference
among the three different subspecies and some
specific signatures.
the vNtR analysis also detected five clusters
encompassing samples from different patients and in
some cases attending different CF centers.
In conclusion, a further assessment of the real
discriminatory power of this panel of vNtR loci is
required in order to confirm the ability of this technique
to detect person-to-person transmission and to avoid
wrong clustering due to insufficient variability of the
markers taken into account.
51
ABSTRACTS OF POSTER PRESENTATIONS (P)
P7
P8
inhA AND katG MuTATIONS ASSOCIATED
WITh ISONIAZID RESISTANCE IN A CANADIAN
PROVINCE
Sara Christianson1, Meenu Sharma1, David
Long2, hong Zhou2, Zhiwei Gao2, Courtney
heffernan2, Richard Long2, joyce Wolfe1
1
Public Health Agency of canada
2
university of Alberta
INVASTIGATION OF PRIMARY
ANTITuBERCuLOSIS DRuG RESISTANCE
uSING BACTEC MGIT 960 SYSTEM IN
MYCOBACTERIuM TuBERCuLOSIS COMPLEx
STRAINS
Ilhan Afsar1, Yusuf Saglam1, Selcuk kaya1,
Mustafa Demirci1
1
ikcu Ataturk training and research Hospital
Background: Isoniazid (INH) resistance in
Mycobacterium tuberculosis (MtB) has important
implications in diagnosis, prophylaxis and treatment of
tuberculosis disease (tB). In 2013, Canada reported
drug susceptibility results for 1397 MtB complex
isolates. INH resistance was reported in 6.7% of
cases. This study investigates the prevalence and
distribution of gene mutations in the katG gene and
inhA promoter region in INH-resistant isolates from
Alberta, Canada.
Methods: All adult (age >14 years) culture-positive,
INH-resistant pulmonary cases of MtB in Alberta,
Canada from 1991-2010 were surveyed for this
study. In total, 97 INH- resistant MtB strains and
45 susceptible strains were included. The resulting
142 strains were tested for INH resistance at critical
concentrations (CC) of 0.1 µg/mL and 0.4 µg/mL.
Strains were also tested for mutations in the katG
gene and inhA promoter region.
Results: of the INH-resistant strains tested, 21 were
resistant at only the lower CC of INH and 76 strains
were resistant to the higher CC of INH. No mutations
were seen in the 45 sensitive strains. Of the strains
with low level resistance, 15/21 (71%) had mutations
in the inhA promoter region. The most common inhA
mutation in these strains was -15 C to t (n=11). one
mutation was recorded in the katG gene of the lowlevel resistant strains, but was accompanied by an
inhA mutation. Two of the high-level resistant strains
had inhA mutations (-15 C to t) and 62 had katG
mutations, the most common of which was Ser 315
thr (n=53). ten strains (13%) resistant at the higher
INH CC had no inhA or katG mutations. All katG and
inhA mutations identified in this study have been
previously associated with INH resistance.
Conclusion: Mutations in the katG gene and inhA
promoter were able to detect 83% of INH-resistant
cases in this study population. Mutation rates for
genes associated with INH resistance in Alberta are
consistent with those reported in the united States
and elsewhere.
Tuberculosis is a disease that stil keeping its
importance. It is first disease in infectious deaths in
all deaths among the world. In our study it is targeted
to determine primer antituberculosis drug resistance
of 59 Mycobacterium tuberculosis complex strains
that have diagnosis of primer tuberculosis the using
BACtEC MGIt 960 system in Izmir Atatürk training
and research Hospital Departmant of Microbiology in
2010-2014.
All patients were newly diagnosed tuberculosis and
the mean patients age the strains were isolated
were 54.2 (18-88 age) the strains were isolated
consequently % 55.9 from sputulum ,%11.9 from
surgery materials,%10.1 from urine , %8.5 from
biopsy materials, %3.4 from bronchoalveolar lavage
fluid, %3.4 from the peritoneal fluid, %3.4 from join
fluid , %1.7 from the fastinggastric fluid and %1.7
from the wound.
In our study,54 (%91,5) strains were sensitive to the
all primer antituberculosis drugs, 5 (%8.5) were found
to be resistant to one or more antituberculosis drugs.
Resistances of the strains that working sensitivities
to only streptomycin, isoniazid and rifampycin were
detected to be 1(%1.7), 1(%1.7), 2(% 3.4) respectively.
Resistance for ethambutol wasn’t detected. total
resistance for single drug was detected to be 4 (%
6,8). the strains of multidrug resistance (resistance for
rifampin and isoniazid) and any two drugs resistance
were not detected. The strain of three drugs resistance
was detected to be 1 (%1,7). General resistance of
strains to streptomycin, isoniazid, rifampycin and
ethambutol were detected to be 2 (% 3.4), 2 (% 3.4),
2 (% 3.4), 1 (% 1.7).the resistance rates of strains
have been detected at below of turkey’s resistance
rates
The aim of our study is to estimate the resistance
profiles of 4 major primer antituberculosis drugs
for primer diagnosed patients of tuberculosis that
detected in İzmir.
52
P15
the sensitivity and specificity were 95% and 98% for
slides and Genocard, 96% and 98% for FtA card and
95% and 93.46% for samples stored in ethanol.
Conclusion: The four transport and storage
supports showed a good sensitivity and specificity
for the detection of resistance to RIF and INH in M.
tuberculosis strains. Evaluation of the potential use of
these four systems to detect RIF and INH resistance
from direct sputum samples is under evaluation.
PERFORMANCE OF FOuR TRANSPORT AND
STORAGE SuPPORTS FOR MOLECuLAR
DETECTION OF MuLTIDRuG RESISTANT
TuBERCuLOSIS
Marie Sylvianne Rabodoarivelo1, Belen
Imperiale2, Angela Brandao3, Parveen kumar4,
Sarman Singh4, Lucilaine Ferrazoli3, Nora
Morcillo2, Voahangy Rasolofo5, juan Carlos
Palomino6, Peter Vandamme6, Anandi Martin6
1
unité des Mycobactéries, institut Pasteur de
2
Hospital Dr. cetrángolo, buenos Aires,
Argentina
3
instituto Adolfo lutz, Núcleo de tuberculose e
Micobacteriosis, sao Paulo, brazil
4
All india institute of Medical sciences, Delhi,
india
5
institut Pasteur de Madagascar
6
IN VITRO ANTI-MYCOBACTERIAL ACTIVITY
OF SELECTED MEDICINAL PLANTS AGAINST
MYCOBACTERIuM TuBERCuLOSIS AND
MYCOBACTERIuM BOVIS STRAINS
Abdella Gemechu1, Mirutse Gidaqy2, Gobena
Ameni2, Adane Worku2
1
college of Health & Medical sciences, Haramaya
university
2
Aklilu lemma institute of Pathobiology, Addis
Ababa university
Background: Detection of drug resistant tuberculosis
(tB), particularly in low-resource countries, is often
hampered by the limitation of transport and storage
of samples from remote locations to the reference
laboratory. Consequently, the treatment of tB usually
starts without the results of drug susceptibility testing
(DSt) being known or treatment gets delayed.
Objective: The aim of this study was to evaluate the
performance of four supports enabling the transport
and storage of samples to be used for molecular
detection of drug resistance using the commercial
Genotype MtBDRplus.
Material and methods: We performed a multicenter
retrospective study with the following participating
sites: Institute Pasteur of Madagascar (IPM); All India
Institute of Medical Sciences (AIIMS), Delhi, India;
Hospital Dr. Cetrángolo, Buenos Aires, Argentina
and Instituto Adolfo Lutz, Sao Paulo, Brazil. Fifty
Mycobacterium tuberculosis strains were selected at
each site (25 MDR and 25 susceptible) to evaluate
the DNA extraction and detection of resistance
to rifampicin (RIF) and isoniazid (INH) using the
Genotype MtBDRplus from samples stored on
slides, on filter cards, such as the FtA card (Whatman
International Ltd) and the GenoCard (Hain Lifescience,
Nehren, Germany), and on samples stored in ethanol.
The performance of each system was assessed
separately by calculating sensitivity and specificity,
compared to the results obtained by DST performed
by the proportion method on Löwenstein-jensen (Lj)
medium or the MGIt960 system.
Results: From all sites, the sensitivity and specificity
for detection of resistance to RIF was 95% and
100%, respectively, for DNA extracted from slides
and Genocard; 96% and 100% for DNA extracted
from FtA card and 96% and 98% for samples stored
in ethanol. For the detection of resistance to INH,
Background: tuberculosis (tB) is a global burden with
one – third of the world ’ s population infected with the
pathogen Mycobacterium tuberculosis complex and
annually 1.4 million deaths occur due to the disease.
This high incidence of infection and the increased rate
of multi-drug resistant and extensively-drug resistant
strains of the organism further complicated the
problem of TB control and have called for an urgent
need to develop new anti-TB drugs from plants. In this
study, the in vitro activity of root of Calpurnia aurea,
seeds of ocimum basilicum, leaves of artemisia
abyssinica, Croton macrostachyus, and Eucalyptus
camaldulensis were evaluated against M.tuberculosis
and M. bovis strains.
Methods: Five Ethiopian medicinal plants, root of
Calpurnia aurea, seeds of ocimum basilicum, leaves
of artemisia abyssinica, Croton macrostachyus,
and Eucalyptus camaldulensis used locally for the
management of TB. They were investigated for in
vitro antimycobacterial activity against M. tuberculosis
and M. bovis strains. 80% methanolic extracts of the
plant materials were obtained by maceration. The
antimycobacterial activity was determined using 96
wells of microplate with the help of visual Resazurin
Microtiter Assay.
Results: the crude 80% methanolic extracts of
the root of C. aurea, seeds of o. basilicum, and
leaves of a. abyssinica, C. macrostachyus, and E.
camaldulensis had anti-mycobacterial activity with
minimum inhibitory concentration (MIC) ranging from
6.25 – 100 μ g/mL. the MIC of 80% methanol extracts
in the order mentioned above ranged 25-100 μ g/ml
and 12.5-75 μ g/mL, 25 – 100 μ g/mL and 25 – 50 μ
g/mL, 6.25-50 μ g/mL and 12.5-50 μ g/mL, 12.5-100
μ g/mL and 18.25-50 μ g/mL and 6.25-50 μ g/mL and
12.5-50 μ g/mL, respectively for M. tuberculosis and
M. bovis strains.
P19
53
Conclusions: The results support the local use of
these plants in the treatment of TB and it is suggested
that these plants may have therapeutic value in the
treatment of tB. However, further investigations are
needed on isolating chemical constituents responsible
for eliciting the observed activity in these plants.
keywords: Antimycobacterial activity, Medicinal
plants, MIC, REMA, M. tuberculosis & M. bovis
strains, Ethiopia
P102
PREVALENCE OF BEIjING GENOTYPE
AMONG MDR ISOLATES OF MYCOBACTERIuM
TuBERCuLOSIS FROM POLAND
Monika Kozińska1, Ewa Augustynowicz Kopeć1,
Sylwia Brzezińska1, Agnieszka Napiórkowska1,
Anna Zabost1
1
research institute of tuberculosis and lung
Diseases
the Beijing family of Mycobacterium tuberculosis
represents about 50% of all tB strains in East Asia
and at least 13% of strains worldwide. First reported
in East Asian countries where they are highly endemic
and later found to be disseminated throughout the
world, Beijing family strains are predominant in the
former uSSR countries, especially in Russia. With
an intrinsic advantage over other M. tuberculosis
genotypes in terms of virulence, Beijing lineage
strains are notably associated with multiple drug
resistant (MDR) and extensively drug resistant (xDR).
In Poland the first incident of Beijing tB was found in
2000.
Materials and Methods: A total of 81 MDR M.
tuberculosis isolates, collected at the National
tuberculosis and Lung Diseases Institute in Warsaw,
were included in the study. The isolates were
recovered from 81 unrelated pulmonary TB patients
(66 men and 15 women) from across Poland. these
patients represented all bacteriologically confirmed
MDR tB cases reported in Poland in 2012-14.
Primary isolation, culturing and species identification
were carried out by standard mycobacteriological
procedures, essentially as described elsewhere. Drug
susceptibility testing against first and second-line
anti-tB drugs was performed using the 1% proportion
method on Löwenstein–jensen (Lj) medium and
molecular tests (Genotype MtBDR, Hain). All
isolates were characterized by spoligotyping with a
commercially available kit (Isogen Bioscience Bv,
Maarssen, the Netherlands).
Results: thirty two of spoligopatterns were identified.
of the 81 isolates, 55 (68%) were located in 6 clusters
of which the largest – Beijing - accounted 36 (44%)
isolates. Among non-Beijing clusters we found St53
(7 isolates/9%), St891 (5/6%), St65 (3/4%), St877
(2/2.5%) and St39 (2/2.5%). the remaining 26 (32%)
isolates did not show clustering. In population with
Beijing tB we observed 8 (10%) patients with xDR
54
and 14 (17%) with pre-xDR tB.
Conclusions: Spoligotyping of 81 MDR-tB isolates
revealed a high number (44%) of Beijing genotype
strains among MDR patients in Poland. the specific
reason for the high prevalence of this genotype is
unknown, but explanations include more recent
transmission of Beijing MDR strains and higher
virulence of these isolates. This observation suggests
that all MDR strains in Poland should be analyzed by
spoligotyping – to control spread of Beijing tB.
P113
MOLECuLAR INVESTIGATION OF MuTATIONS
ASSOCIATED WITh RIFAMPICIN AND ISONIAZID
RESISTANCE IN CLINICAL ISOLATES OF
IN MODENA, ITALY
Giulia Fregni Serpini1, Sara Tagliazucchi1,
Francesca Frascaro1, Antonella Grottola1, Nadia
Nanni1, Giulia Forbicini1, Rita Magnani1, William
Gennari1, Anna Fabio1, Fabio Rumpianesi1,
Monica Pecorari1
1
Microbiology and Virology unit, university
Hospital Policlinico of Modena, italy
tuberculosis is a major cause of morbidity and
mortality, as well as in developing countries, even in
industrialized countries. Furthermore, the emergence
of drug-resistant Mycobacterium tuberculosis
complex (MtBC) strains is considered a real threat to
achieving tuberculosis control.
The aim of this study is to investigate the gene
mutations conferring resistance to rifampicin (RIF)
and to isoniazid (INH) in MtBC strains isolated in
Modena since 2007 to 2014.
Phenotypic drug susceptibility test (DSt) detected 56
resistant strains (15 were MDR and 41 were resistant
to isoniazid) and 56 susceptible strains. We analyzed
all 112 strains with the Genotype MtBDRplus kit
and by sequencing to detect mutations conferring
resistance to RIF (rpoB gene) and INH (katG gene,
inhA gene and inhA promoter).
The results of 56 phenotypic DST susceptible strains
were confirmed by both molecular methods while, for
the 56 resistant strains, only 50 were confirmed as
resistant to both molecular methods. Among the 41
isolates resistant to INH, the most frequent mutation
detected by the Genotype MtBDRplus and by
sequencing were S315t in katG gene (56%) followed
by -C15T in inhA promoter (34%). In addition, a rare
mutation -G102A in inhA promoter in 1 strain and the
katG polymorphism R463L in 17 strains (alone or in
association with S315t) were detected by sequencing.
MtBDRplus found the lack of the rpoB WT7 probe in
2 out of 41 INH resistant strains indicating the RIF
resistance; this result was confirmed by sequencing
showing the rpoB H526L mutation. Moreover, in 1 of
these 2 strains the rare mutations rpoB S469L and
inhA S94A/v78A were found by sequencing. Six of 41
isolates phenotypically resistant to INH appeared to
be Wt with both molecular methods. out of 15 MDR
strains, the most common mutations detected by both
molecular methods in inhA promoter and in katG gene
were respectively -C15t (26,6%) and S315t (93,3%)
while in rpoB gene the most frequent mutation was
S531L (93,3%) followed by the D516v (13,3%).
the sequencing showed the katG polymorphism
R463L associated with the katG S315T mutation in
4 strains; another MDR strain without katG detected
mutation by MtBDRplus showed in katG gene the
polymorphism R463L and the rare mutation E289G
described elsewhere in a resistant strain. In 1 strain,
the molecular kit highlighted, in addition to S531L, the
rpoB H526D mutation not found by sequencing.
We can conclude that the occurrence of mutations
found in the study is in agreement with literature data.
the sequencing has highlighted rare mutations in
resistant strains: rpoB S469L never described in the
literature to our knowledge, inhA S94A/v78A and katG
E289G independently detected by other authors.
P114
COMPLETE GENOME SEquENCE OF ThE
MuLTIDRuG-RESISTANT BEIjING-LIkE STRAIN
MYCOBACTERIuM TuBERCuLOSIS 323
uSING ThE PacBio REAL-TIME SEquENCING
PLATFORM
juan Germán Rodríguez1, Camilo Pino2, Andreas
Tauch3, Martha I Murcia1
1
Grupo Micobac-un, Departamento de
Microbiología, Facultad de Medicina,
universidad Nacional de colombia
2
Grupo biolisi, Facultad de ingeniería,
universidad Nacional de colombia, sede bogotá
3
institut für Genomforschung und
systembiologie, centrum Für biotechnology
(cebitec), universität bielefeld
Strains of the Mycobacterium tuberculosis Beijing
lineage have become successful pathogens worldwide
due to their capacity to spread in a population. In
some cases these strains have been associated
with multidrug-resistant tuberculosis outbreaks. In
Colombia, the Beijing lineage was reported since
1998 and recent evidence suggests that strains of
this lineage have an increased incidence and are
associated with multidrug-resistant tuberculosis
outbreaks. We therefore sequenced the complete
genome of a M. tuberculosis Beijing-like strain with the
internal nomenclature “isolate 323” to gain insights into
its genetic characteristics. The spoligotyping method
was used to confirm the assignment of isolate 323
to the Beijing-like lineage of M. tuberculosis. It was
classified as Spoligotype International type SIt190.
Genomic DNA was extracted from isolate 323 following
the CEtAB method. the DNA was sequenced using
the PacBio RS II sequencer. the hierarchical genome
assembly process from the Pacific Biosciences
SMRt analysis tool-kit was used to obtain a de novo
assembly. The results showed an assembly with a
single contig and a sequence length of 4,411,216 bp
with an average depth of coverage of 298.65 ×. The
N50 contig length was 8,627 bp and the mean read
length was 5,971 bp. the assembled sequence was
annotated using the GenDB annotation system for
prokaryotic genomes. the genome sequence has a
mean G+C content of 65.6%, 4,237 protein-coding
regions, 45 tRNA genes, and 3 rRNA genes. taking
the genome of M. tuberculosis H37Rv as a reference
sequence, the assembly has an identity of 99.88%,
with 3,507 total variants, comprising 2,143 single
nucleotide polymorphisms (SNPs), 207 multiple
nucleotide polymorphisms, 588 insertions, and 569
deletions. Among the SNPs 1787 were detected
in coding regions, of which 743 are synonymous,
1,026 missense and 18 nonsense substitutions.
SNPs known to confer resistance to rifampin (S450L
in rpob), isoniazid (S315t, R463L in katG and
D229G in accD6), ethambutol (M306v in embb),
pyrazinamide (T142A in pnca), and streptomycin
(K43R in rpsL, E92D in gidb) were observed and
confirm the multidrug-resistant phenotype of isolate
323. Moreover, a novel mutation was observed in
the embC gene (del C2972bp) coding for ethambutol
resistance. Regarding second-line antituberculous
drugs, a known mutation in the gyra gene (D94G)
and the novel mutations E21q and D668G were
detected. SNPs known to confer resistant to amikacin/
kanamycin/capreomycin (A1401G in rrs) and a
novel mutation in the eis gene (del CA804bp) were
observed, suggesting that this strain is extensively
drug-resistant. this is the first report of whole-genome
sequencing of a multidrug-resistant M. tuberculosis
Beijing-like isolate circulating in Colombia and we
demonstrate that the PacBio sequencing technology
is a reliable method for generating finished microbial
genomes of M. tuberculosis isolates.
P129
POPuLATION STRuCTuRE OF MuLTIDRuGRESISTANT MYCOBACTERIuM TuBERCuLOSIS
ISOLATES
Claudia Llerena Polo1, Martha I Murcia2, juan
Germán Rodríguez2, julio Guerra2
1
instituto Nacional de salud
2
Introduction and objective: In Colombia the
prevalence of multidrug resistant tuberculosis (MDRtB), has slowly but constantly increased since 2001.
Each year there are about one hundred cases of MDRtB, and 250 cases of drug-resistant tB (resistance
to any drug). Most of these cases are diagnosed in
Antioquia and valle del Cauca departments. the
Colombian National Institute of Health confirmed 36
55
xDR-tB. the objective of this study was determined
the population structure of Mycobacterium tuberculosis
complex strains in a cohort of MDR-tB Colombian
patients.
Methods: A total of 201 MDR Mycobacterium
tuberculosis complex strains from patients isolated
during 2012 – 2013, in 24/33 departments of
Colombia, were studied. Resistance profiles were
confirmed using BACtEC-MGIt 960 and all strains
were studied by second line susceptibility by the
same method. The strains are being genotyped by
spoligotyping.
Results: of the 201 tB patients, 165(82%) were
previously-untreated second line anti-TB drugs and
the remaining 36(18%) were previously-treated
cases, 7(3,4%) of them were xDR-tB. the various
spoligotyping-based genotypic lineages isolates
were: Latin American & Mediterranean (LAM) n =
73(36,3%); Haarlem, n = 54(26,9%); ill-defined t
group, n =29(14,4%); Beijing, n =22(10,9%); u family,
n=11(5,5%); S, n =5(2,5%) and x clade, n = 5(2,5%).
The lineage distribution of M. tuberculosis showed
that more than strains in Colombia are commonly
found in Latin America.
Conclusion: the most frequely linages founded
in MDR-tB in Colombia‘s strains belong to LAM,
Haarlem and Beijing linages.
Keywords: Mycobacterium tuberculosis, Population
structure, Spoligotyping, MDR-tB, Colombia.
P156
GENOTYPING AuTOMATIZATION: PRELIMINARY
RESuLTS ON FIRST AND SECOND-LINE DRuG
RESISTANCE DETECTION IN M. TuBERCuLOSIS
COMPLEx
harrison Magdinier Gomes1, Bernice klotoe2,
Barbara Molina3, jose Dominguez3, Ali Sajid4,
Maria helena Feres Saad5, Michel kiréopori
Gomgnimbou6, Christophe Sola2
1
France; laboratory of Molecular biology Applied
to Mycobacteria, oswaldo cruz Foundation, rio
de Janeiro
2
institut de biologie intégrative de la cellule
3
servei de Microbiologia, Hospital universitari
Germans trias i Pujol
4
Quaid-i-Azam university
5
institute oswaldo cruz Foundation
6
université Paris-sud-cnrs, institut de Génétique
et Microbiologie, the infection, Genetics,
emerging Pathogens (igepe) team, orsay,
France; centre Muraz, bobo Dioulasso, burkina
Faso
MDR-tuberculosis remains an important threat.
Proportion method is precise and widely used to
detect MDR-tB however results can be obtained at
best in 12 days when using Liquid culture. Innovative
methods based on genetics are needed.
56
Gomgnimbou et al. described “TB-SPRINT” an highthroughput microbead-based method that detects
rpoB, katG, inhA mutations related to resistance to
rifampicin and isoniazid respectively and that produce
a spoligotype pattern. We developed a secondgeneration assay to detect mutations in the embB,
gyra, eis, rrs genes related to drug resistance to
ethambutol, fluoroquinolones, kanamycin respectively
constituting a total of 18 probes (TB-SPRINT ultra).
to reduce laboratory costs, the goal of this new
study was to perform automatization of coupling and
hybridization. three basic steps: beads coupling, mix
building, PCR, hybridization/reading, are routinely
performed manually. We used a semiautomated
process, using two robots: Andrew (AndrewalliancetM
Geneva, Switzerland) (for coupling) and PipetMax®
(Gilson France, villiers le Bel ) for hybridization.
Andrew is a mobile anthropomorphic. PipetMax is a
mixed (mono and multi-channel) pipetting platform
from Gilson. Detection/Reading was done on a
Luminex 200 (Luminex Corp, Austin, texas).
222 M. tuberculosis samples, (124 from Brazil and
98 from Pakistan), were tested for first and second
line drug-resistance mutations. The results show
that the robots performed with the same quality of
results as obtained manually, even though in both
automated systems, we observed loss of microbeads.
These promising results lead us to look in the future
for solutions with higher levels of automatization.
use of magnetic microbeads and MagPix® could
also improve bead-assay efficiency. Small reagent
production batches for local MDR-tB diagnostic,
mutation analysis, and epidemiological studies,
using a “personalized and decentralized” molecular
diagnostics model could be pioneered in the future.
P175
PhENOTYPIC FLuOROquINOLONE
SuSCEPTIBILITY TESTING IN M.
TuBERCuLOSIS COMPARING REMA, MGIT 960
AND SOLID MEDIuM
Nele Coeck1, Bouke de jong1, Leen Rigouts2
1
institute of tropical Medicine, Department of
biomedical science, Mycobacteriology unit,
university of Antwerp, belgium
2
institute of tropical Medicine, Faculty of
Pharmaceutical, biomedical and Veterinary
sciences, univeristy of Antwerp, belgium
While the fourth generation of fluoroquinolones (Fqs)
has shown greater effectiveness for treatment of
multidrug-resistant tuberculosis (MDR-tB) compared
to the older Fqs, the level of in vitro cross-resistance
between the Fqs using different drug susceptibility
testing
formats,
remains
under-documented.
therefore, we investigated cross-resistance between
various generations of Fqs, by comparing MIC
determination for ofloxacin (ofx), levofloxacin (Lvx),
moxifloxacin (Mfx), and gatifloxacin (Gfx) in REMA
and on Lj medium (as the golden standard), with
ofx susceptibility testing in the Bactec MGIt 960
system, in addition to DNA sequencing of the gyra
and gyrb genes. A total of 93 M. tuberculosis isolates
were included, comprising 72 previously identified as
ofx-resistant and 21 ofx-susceptible on Middlebrook
7H11 (proportion method, cut-off 2 µg/ml), as per
routine testing. For all media, a cut-off of 2 µg/ml for
ofx, 1 µg/ml for Lvx, and 0,5 µg/ml for Mfx and Gfx
was applied.
Among the ofx-resistant isolates on Lj, 98%, 96%
and 87% were found cross-resistant to Lvx, Mfx and
Gfx respectively. Around 62% of the isolates with
elevated MICs for ofx, Lvx, Mfx and Gfx on Lj showed
one or more high-confidence mutation, with 94Gly
and 90val as the most common ones. Remarkably,
33% of the isolates showing an ofx MICs of >2 µg/ml
on LJ harboured no gyra and gyrb mutations.
In REMA, only 48%, 49%, 33% and 9% of the ofxresistant isolates on Lj showed resistance to ofx, Lvx,
Mfx and Gfx respectively. Among the ofx-resistant
isolates with high-confidence mutations, we achieved
a ofx sensitivity rate of 56% in REMA and 94% in
MGIt 960. Lowering the cut-off for ofx towards 1 µg/
ml in REMA increased the sensitivity towards 94%.
In conclusion, these data confirm cross-resistance
between the various Fqs, although new generations
of Fqs had systematically lower MICs. In addition,
the high rate of probable false-susceptible results
in REMA suggests the need for lowering the current
applied cut-offs of Fqs in REMA, however a more
extensive validation experiment is warranted.
Remarkably, a high proportion of Fq-resistant isolates
harboured wild-type gyra and gyb genes which could
be attributed to undetected heteroresistance or the
involvement of alternative resistance mechanisms,
such as efflux pumps.
P191
PhENOTYPIC AND GENOTYPIC INVESTIGATION
OF MuLTIDRuG RESISTANCE (MDR) AND
ExTENSIVELY DRuG RESISTANCE (xDR) IN
MYCOBACTERIuM TuBERCuLOSIS STRAINS
ISOLATED FROM CLINICAL SAMPLES IN
CukuROVA REGION, TuRkIYE
Togrul Nagiyev1, Emel Yarar1, Begum kayar2,
Farhad kohansal1, Gulfer Yakici1, Taylan Bozok1,
Fatih koksal1
1
Department of Medical Microbiology, Medical
Faculty, cukurova university, Adana, turkey
2
tropical Disease research and Application
center, cukurova university, Adana, turkey
occurence of the Extensively Drug-Resistant (xDR)
strains by improving resistance to second-line antituberculosis (anti-tB) drugs of multidrug-resistant
(MDR) Mycobacterium tuberculosis complex (MtBC)
strains brings about one of the most important human
health threats. We aimed to determine the first- and
second-line anti-TB drug resistance patterns of
MtBC strains isolated from Cukurova region, turkiye
by phenotypic and genotypic methods.
A total of 703 MtBC isolates obtained from 414 tBsuspected patients from 19 hospitals/dispensaries
in Cukurova region throughout 2012 were enrolled
in the study. First- and second-line anti-tB drug
resistance patterns of the isolates were phenotypically
determined by BACtEC MGIt 960 automated system
and Lowenstein Jensen agar proportion susceptibility
methods, respectively. Genotypic verification of the
presence of MtBC and the MDR/pre-xDR/xDR was
performed on cultures of MDR-tB isolates by multiplex
Real-Time PCR assay using the novel Anyplex™ II
MtB/MDR/xDR Detection kit (Seegene).
Resistance to at least one first-line anti-tB drug
and MDR pattern were phenotypically determined
in 193 (27.5%) and 47 (6.7%) of the MtBC isolates
belonging to 112 (27.1%) and 21 (5.1%) patients,
respectively. All of the 47 MDR-MtBC isolates were
verified as MtBC however 37 (78.7%) of them verified
as MDR-MtBC by Real-time PCR testing. thus
sensitivity and specifity of the Real-time PCR assay
in MDR detection was 78.7% and 100%, respectively
comparing with phenotypic methods. two pre-xDR
strains, one fluoroquinolone- and one injectable
drug-resistant isolates were detected, however xDRMtBC was not detected with both of phenotypical and
genotypical methods. It was observed that results of
the phenotypic and genotypic investigations were
very consistent,
In conclusion, it was suggested that the new multiplex
Real-Time PCR assay is usable and reliable method
for detection of MtBk, MDR and xDR in several
hours.
key Words: Agar proportion susceptibility method,
BACtEC MGIt 960, Extensively Drug Resistance
(xDR), Multidrug Resistance (MDR), M.tuberculosis
complex (MtBC), Real-time PCR.
P192
INVESTIGATION OF EPIDEMIOLOGICAL
ChARACTERISTICS AND MOLECuLAR
MEChANISMS OF MOxIFLOxACIN RESISTANCE
IN MYCOBACTERIuM TuBERCuLOSIS STRAINS
ISOLATED FROM PuLMONARY TuBERCuLOSIS
PATIENTS
Taylan Bozok1, Begum kayar2, Gulfer Yakici1,
Mahdi Marzi1, Togrul Nagiyev1, Emel Yarar1, Firat
karsli1, Fatih koksal1
1
2
57
Objective: In this study, it is aimed to determine
the moxifloxacin sensitivities of Mycobacterium
tuberculosis strains isolated in our region and to
specify the regional resistance rates besides to
investigate the relation of phenotypical resistance
with mutations in gyra gene.
Materials and Method: Sequential 100 isolates
that are seen to be non-multi drug resistant and 37
isolates that are determined as multidrug resistant
among Mycobacterium tuberculosis strains isolated
from pulmonary tuberculosis patient’s samples which
sent to Region Tuberculosis Laboratory of Adana
were included in this study. Drug sensitivity rates and
DNA Sequencing of gyra region of these isolates are
compared again.
Findings: Resistance to moxifloxacin is found in
25 of 137 (18,2 %) isolates of which phenotypic
susceptibilities were tested. Resistance rates among
strains which seen to have multidrug resistance and
those which have not multidrug resistance were 17 %
and 21,6 % respectively. Looking at the distribution
according to regions which sent 25 clinical isolates;
it is determined that 10 of isolates were belonged to
Adana, 6 to Gaziantep, 5 to Hatay, 3 to Mersin and 1 to
kahramanmaraş province. Among all isolates included
in study, Single bases mutation was encountered
in 6 samples in total according to DNA sequencing
result of gyra region. The positions of mutations were
identified as Asp94tyr, Asp94Gly, Ala90val, Gly88Ala
and in 2 samples as Asp89Asn. Two of the samples
which mutation is seen were found to be sensitive to
moxifloxacin phenotypically.
Result: At the end of our study it is determined that,
resistance developed to moxifloxacin would not
be secondary in the treatment of tuberculosis. It is
seen that different molecular mechanisms will be
responsible from high resistance and the relation
between resistance and mutations in the analysed
gene is insufficient. It is concluded that research on
moxifloxacin resistance should be continue with wider
case groups and isolates also it will be important
to persistently explain that there is a probability of
resistance development in a short time to these group
of drugs and it is required to use them more limited in
non-spesific indications.
keywords: gyra, Moxifloxacin, Mutation, Mycobacterium tuberculosis, Proportion.
P193
FLuROquINOLONE hETERORESISTANCE
Leen Rigouts1, Mirjam Schats2, Nele Coeck2,
Bouke de jong2
1
institute of tropical Medicine, Faculty of
Pharmaceutical, biomedical and Veterinary
sciences, univeristy of Antwerp, belgium
2
institute of tropical Medicine, Department of
biomedical science, Mycobacteriology unit,
univeristy of Antwerp, belgium
58
While the phenomenon of heteroresistance is known
to occur in clinical isolates and is not unique to the
fluoroquinolone class of antibiotics (Fqs), the limit
of detection (LoD) using different phenotypic and
genotypic assays, as well as the clinical relevance of
heteroresistance remain poorly documented.
In this study we explored the LoD to detect Fq
heteroresistance in experimentally mixed Fq-resistant
and Fq-susceptible M. tuberculosis strains by
minimal inhibitory concentration (MIC) determination
of gatifloxacin, Genotype MtBDRsl, gyra Sanger
sequencing and digital PCR.
Starting from a 1 mg/ml bacterial suspension of
a pan-susceptible M. tuberculosis strain (S; wildtype gyra; ItM 011738) and its in vitro selected
Fq-resistant daughter strain (R; 94Gly mutant; ItM
130481), the following mixtures of the S- and R-strain
were prepared (S/R): 0/100, 1/99, 5/95, 10/90, 20/80,
30/70, 40/60, 50/50 and 0/100. Mixture preparation
was repeated 3 times on different days to generate
biological triplicates.
Each ‘fresh’ mixture was inoculated on Middlebrook
7H11 medium at 5 different concentrations (0,25 -0,51,0-2,0 and 4,0 µg/ml) of gatifloxacin, followed by
incubation at 37°C at 5%Co2 for 4 weeks. In addition,
300 µl of each mixture was added to 400 µl 1*tE
buffer, heat killed for 10 minutes at 100°C and stored
at -20°C for molecular assays.
the 100%-susceptible suspension had a gatifloxacin
MIC < 0.25 µg/ml, the 1%-resistant suspension
showed an MIC of 0.25 µg/ml, whereas all remaining
heteroresistant mixtures yielded an MIC of 0.5 µg/ml .
Among the molecular assays tested to date, MtBDRsl
was the most sensitive, detecting a 5% resistant subpopulation, and Sanger sequencing identified 20%
resistance. Results from deep sequencing and digital
PCR are pending.
to investigate the clinical relevance of Fq
heteroresistance,
we
manually
re-analysed
chromatograms of pre-treatment isolates obtained
from an MDR-tB cohort with documented
gatifloxacin-based treatment outcome. Among 117
re-inspected sequences, 38 were confirmed as
gyra mutant and 5 as mutant/wild type mixtures as
detected by the standard software. Among the 74
initially identified as Wt, manual inspection clearly
revealed double peaks - albeit below the detection
limit of the software - in 4 isolates, whereas for 70 no
indication of heteroresistance was observed, yielding
a heteroresistance level of 7.7% (9/117). In ongoing
analysis, these results are linked to MICs, treatment
outcome, as well as digital PCR and deep sequencing
data.
P196
MuTATION ANALYSIS OF MYCOBACTERIAL
RPOB GENES AND EVALuATING MICS
FOR RIFAMPIN IN MYCOBACTERIuM
TuBERCuLOSIS STRAINS ISOLATED
FROM SPuTuM SAMPLES OF PuLMONARY
TuBERCuLOSIS PATIENTS
Begum kayar1, Taylan Bozok2, Adnan Atilgan2,
Fatih koksal2
1
2
Resistance to rifampicin (RIF) cause a serious and
significant threat to tuberculosis control. Control of
drug resistant tuberculosis relies proper treatment of
drug resistant cases and rapid diagnosis.
Molecular
characterization
RIF-resistant
M.
tuberculosis clinical strains of different origins can
generate information useful for developing rapid
molecular diagnostic methods that are widely
applicable.
The aim of this study was to investigate the molecular
epidemiology of rifampicin resistant Mycobacterium
tuberculosis strains isolated in Cukurova Region,
Southern Turkey. We evaluated the mutations in a
329 base pairs located in 481-589 codons of the rpoB
gene by automated sequencing of 50 strains. In 50
rifampicin resistant strains, Mutations were identified
in 98% strains. No mutation was detected in the
(%2) 1 resistant strains. the most common mutation
(51,02%) was Ser531Leu.
overall, 15 different mutations affecting 12 codons
were identified. the codon numbers of the most
frequently encountered mutations were 531 (65,30%),
516 (10,20%), and 526 (6,1%). It was concluded that
the results from this study can serve as a basis for
monitoring molecular epidemiology of drug resistant
M. tuberculosis strains isolated in our region. LJ
proportions cultured. Study is ongoing.
keywords: Drug resistance, Mycobacterium
tuberculosis,rpoB gene, sequencing analyse, MIC
values.
P203
INCIDENCE OF DRuG RESISTANT
TuBERCuLOSIS IN RuRAL hAITI: A
DESCRIPTIVE ANALYSIS
R. justin May1, Md. Siddiqur Rahman khan1,
Madsen Beau de Rochars1, Michael Lauzardo1
1
university of Florida
Incidence rates of tuberculosis (tB) in Haiti are
comparable to those in East Africa. In 2010, WHo
estimated the prevalence of all forms of active
tuberculosis in Haiti to be 314/100,000. While
resistance to anti-tuberculosis drugs has not been
monitored systematically in Haiti, the WHo has
estimated multi-drug resistance rates of approximately
2.1% in newly diagnosed patients and 12% in persons
who have previously been treated for TB. A more
recent study put the incidence rate in Port-au-Prince
at 2.9% among newly diagnosed patients. there is
a great need to better understand the extent of drug
resistant TB in the rural areas of Haiti.
Methods: Sputum specimens were obtained from 513
patients in a convenience sample with confirmed or
suspected pulmonary TB. All specimens were stained
for acid fast bacilli and cultures were performed using
liquid and solid media. Drug susceptibilities were
performed using MGIt, solid media, and genexpert
(Cepheid).
Results: A total of 606 samples were processed from
513 patients. Ninety (14.8%) of these samples were
smear positive. Mycobacteria were detected in 116 of
these samples of which 106 (91.4%) were identified
as MtB. these were further tested for resistance to
INH and Rif, and 30 (28.3%) of which were confirmed
resistant to at least one of the two drugs and 10
(9.4%) were MDR.
Limitations: Liquid media was unavailable to our
laboratory for several months during the study period.
Had we had access to a better supply of liquid media,
our recovery could be significantly higher. Even
though our laboratory is capable of molecular tests
such as Hain and Cepheid, a reliable and practical
solution for getting the reagents into the country has
not been solidified. the samples were obtained from
a convenience sample of patients presenting for care
at a clinic providing medical services.
Conclusion: It is clear that there is a significant
and possibly worsening MDRtB epidemic in Haiti
as the incidence in our rural sample is three times
what has been recently found in an urban setting. A
more deliberate analysis of the true incidence of drug
resistance is needed to better guide programmatic
interventions.
P214
ROLE OF PuTATIVE EFFLux GENE Rv0194
IN DRuG RESISTANCE IN MYCOBACTERIuM
TuBERCuLOSIS
Anshika Narang1, Astha Giri1, Mridula Bose1,
Mandira Varma-Basil1
1
Dept. of Microbiology, Vallabhbhai Patel chest
institute, university of Delhi
Mycobacterium tuberculosis is one of the most
successful pathogens worldwide. Treatment of
tuberculosis (tB) has become very difficult because
of the emergence of multiple drug resistant strains.
Resistance to anti-TB drugs has mainly been
attributed to spontaneous mutations. However, a
proportion of drug resistant isolates have been
observed without characteristic mutations. The
59
lack of information on drug resistance in these isolates
has led to the possibilities of existence of alternative
mechanisms of drug resistance such as drug efflux
pumps in M. tuberculosis.
We investigated the role of a putative efflux pump
gene Rv0194, a member of ABC transporter family in
conferring drug resistance to isolates of M. tuberculosis
obtained from patients of pulmonary tuberculosis.
the study was performed on six Rifampicin (RIF)
susceptible and four Multidrug resistant (MDR) clinical
isolates of M. tuberculosis. Isolates were exposed to
subinhibitory concentrations of RIF followed by Real
Time Analysis using housekeeping genes siga and
rrs as normalizers. Both drug susceptible (n=2) and
MDR (n=3) clinical isolates showed upregulation in
the expression of Rv0194 in comparison with siga and
rrs. Additional 2 drug susceptible isolates and H37Rv
showed upregulation only in comparison with siga.
Cloning and overexpression of Rv0194 in H37Rv
did not reveal any change in Minimum inhibitory
concentration (MIC) of RIF. Sequencing of the RIF
resistance determining region (RRDR) of the isolates
showed the presence of mutations at codon 511 in
one MDR isolate and codon 531 in two MDR isolates.
The drug susceptible isolates lacked mutations in
this region. Interestingly, one of the drug susceptible
isolate overexpressing Rv0194 in comparison with
both siga and rrs was found to be low level resistant
when its MIC was performed by Microplate Alamar
Blue Assay, even though no mutation was found in
its RRDR.
Our results suggest that Rv0194 though not directly
involved in drug resistance in M. tuberculosis may be
upregulated as an initial response to drug stress.
P215
DRuG-RESISTANT TuBERCuLOSIS IN CROATIA,
2010-2014
Mihaela Obrovac1, Ljiljana Zmak1, Vera katalinicjankovic1
1
croatian National institute of Public Health
Mycobacterium tuberculosis still represents a
serious cause of morbidity and mortality worldwide.
tuberculosis (tB) caused by resistant M. tuberculosis
strains poses additional threat as it requires prolonged
and costly treatment with higher mortality rates then
tB caused by sensitive strains. In Croatia today, there
are approximately 500 TB patients per year and drug
sensitivity testing is performed for all bacteriologically
positive patients. Although the percentage of resistant
tB is low, it is important to detect and follow these
patients. The aim of this study was to determine
resistance patterns and genetic lineages of resistant
M. tuberculosis strains circulating in Croatia during a
five year period, between 2010 and 2014. Among
2384 bacteriologically confirmed patients, a total of 88
(3.69%) resistant M. tuberculosis strains (1 strain per
tB patient) were identified. All isolates were genotyped
60
using 15-locus mycobacterial interspersed repetitive
units – variable number of tandem repeats (MIRuvNtR) analysis. M. tuberculosis global lineages were
determined by comparison with MIRu-vNtRplus on
line tool. Isoniazid and rifampicin resistance conferring
mutations were determined using GenoType
MtBDRplus assay (Hain, Germany). the majority of
tested isolates (75%) belonged to the Euro-American
global lineage. The most prevalent sub-lineages were
Haarlem (60.22%), followed by uganda I&II (6.82%)
and Latin American – Mediterranean (3.41%). the
most common resistance pattern was monoresistance
(80.68%), to streptomycin, isoniazid and rifampicin
(30.68%, 48.86% and 1.14%, respectively).
Multiresistant pattern was identified in six (6.82%)
cases. In 10 (11.36%) cases there was isoniazid and
streptomycin resistance and in one (1.14%) case
there was resistance to streptomycin and ethambutol.
Among 59 isoniazid resistant strains, a total of 22 had
resistance conferring mutation in katG gene (S315t),
23 in inha promoter region (C-15t) and 14 had none
of these mutations. In multiresistant strains, the most
prevalent rifampicin resistance conferring mutation
was S531L (in five cases), and in one case there was
a mutation in codones 526-529 (ΔWt7). In conclusion,
in Croatia there is a low number of patients with
resistant strains, but relatively high number of strains
with monoresistance, especially to isoniazid. these
patients have to be monitored with caution, as there
is a possibility of developing resistance to rifampicin
and becoming multiresistant.
P39
P44
IN VITRO ACTIVITIES OF EDP-420 AND EDP322 AGAINST SEVERAL NONTuBERCuLOuS
MYCOBACTERIA
Carolyn Shoen1, Mary Sklaney1, Michael
Cynamon1
1
Vamc, suny upstate Medical university,
syracuse, New york, united states
RNA AND PROTEIN INTERACTING PARTNERS
OF MYCOBACTERIuM SMEGMATIS RNAP
Jiří Pospíšil1, jarmila hnilicová1, Martina
janoušková1, Libor krásný1
1
institute of Microbiology, Academy of sciences
of the czech republic
EDP-322 and EDP-420 are bicyclolides (bridged
bicyclic macrolides). EDP-420 previously was found
to have promising in vitro and in vivo activity against
M. avium complex (MAC). the purpose of the present
study was to evaluate the comparative in vitro activities
of EDP-322, EDP-420 and clarithromycin against M.
abscessus (MA), MAC, and M. kansasii (Mk).
EDP-322, EDP-420, clarithromycin (CLA), and
amikacin (AMk) were obtained from Enanta
Pharmaceuticals
(Watertown,
MA),
Abbott
Laboratories (Abbott Park, IL), and Bristol-MyersSquibb Co. (Princeton, Nj) respectively. these
compounds were dissolved in DMSo at 5 mg/ml prior
to freezing at -200C. The compounds were serially
diluted in 50µl of Middlebrook 7H10 broth (pH 6.6
[7H10 agar formulation with agar and malachite green
omitted]) supplemented with Middlebrook oleic acidalbumin-dextrose-catalase enrichment and 0.05%
tween 80 (MAC), 7H9 broth (Mk), or Mueller Hinton
broth (MA) in polystyrene 96-well round-bottom
microtitre plates. to each well, 50µl of the appropriate
mycobacterial suspension was added to yield a final
concentration of about 1 x 105 CFu/ml. Plates were
incubated at 370 C in ambient air for 7 days (MAC and
Mk) or 5 days (MA) prior to reading.
the MIC50/MIC90 (µg/ml) of EDP-322, EDP-420, and
CLA against Mk isolates (N=22) were 4/8, 1/2, and
0.125/0.125 respectively. the MIC50/MIC90 of EDP322, EDP-420, CLA, and AMk against MA isolates
were 8/64, 2/64, 1/32, and 2/4 respectively.
the MICs (µg/ml) for EDP-322, EDP-420 and CLA
against macrolide naïve MAC isolates (N=13)
were <32, ≤4 and ≤2 respectively. For macrolide
experienced isolates (N=10) with CLA MICs >64 µg/
ml (except for one isolate with an MIC of 64) the EDP322 and EDP-420 MICs were ≤64 µg/ml except for
one isolate which had an MIC >64 µg/ml.
Evaluation of EDP-322 and EDP-420 in murine
models of mycobacterial infection would better define
the potential role of these agents as therapeutics for
mycobacterial infections in humans.
RNA polymerase (RNAP) is the central enzyme of
transcription. Mycobacterium smegmatis RNAP is
composed of core subunits (α2ββ´ω1ω2) and a specific
sigma factor that recognizes the promoter sequence
and allows transcription initiation. RNAP carries out
the polymerization function and its activity is regulated
by various auxiliary factors. These factors can be
both proteins or small RNAs. Here, we will describe
a newly discovered sRNA, termed Ms1 that interacts
with RNAP core in stationary phase. Further, we will
present quantification of protein interaction partners
of mycobacterial RNAP by mass spectrometry from
exponential and stationary phase. thus, this project
contributes to our understanding of the transcription
machinery in mycobacteria, a medicinally important
group of bacteria.
P46
GENE AND GENOME ANALYSIS INDICATES
ThAT ThE STRAIN jS623 IS WRONGLY
CONSIDERED A MEMBER OF ThE SPECIES
MYCOBACTERIuM SMEGMATIS
Maria jesus Garcia1, S. Gola2
1
Facultad de Medicina, universidad Autónoma de
Madrid, spain
2
centro Nacional de biotecnologia (cnb),
Consejo Superior de Investigaciones Cientificas
During a study on the application of whole genome
sequence comparisons to differentiate mycobacterial
species, unexpected differences were found between
the genome of strain JS623 and the genome of strain
mc2155, both considered members of the species
Mycobacterium smegmatis. Both are among the few
strains of that species having a complete genome
sequence available in the data-bases.
Aiming to clarify the memberships of both strains
to that species, we performed a comprehensive
molecular phylogenetic analysis of jS623, mc2155
and other members of the genus.
Single and concatenated gene trees, including the
sequences of the M. smegmatis type strain, showed
that only strain mc2155 and another, different,
61
strain (MkD8) clustered with the type strain. the
molecular phylogenetic analyses suggest that
strain JS623 is a mycobacterium more related to
Mycobacterium moriokaense than to M. smegmatis,
and indicate that it is not a member of this last species,
as was previously believed.
Moreover, according to the standard values accepted
for species delimitation, using three different whole
genome sequence procedures of comparisons, the
parameters of JS623 clearly separate this strain from
the other two strains of M. smegmatis. Percentages of
digital DNA-DNA hybridization were 20.3%; Average
Nucleotide Identity 81.8%, and gene rearrangements
was higher than 94%. All these values indicate a
separation of JS623 from the other M. smegmatis
strains at the genome level.
To avoid future confusions as well as misleading
conclusions, the strain jS623 should be corrected as
Mycobacterium sp. not only in the literature but, even
more importantly, in the database entries. our results
underline the strength of genome comparisons to
identify erroneously classified genome entries.
Acknowledgements: We wish to thank M. Pavelka
for helpful comments on the history of the MkD8
strain and N.S. osório for the communication of
unpublished results on LldD.
Funding resources: International Cooperation CEALuAM and the Spanish Ministry of Health (AES, PI1301218).
P86
NEW GENOME SEquENCE OF A MAP
ShEEP STRAIN: jIII-386 FROM GERMANY
// ANNOTATION AND COMPARISON WITh
PuBLIShED MAP-S AND MAP-C STRAINS
Petra Möbius1, Martin hölzer2, Marius Felder3,
Gabriele Nordsiek4, Marco Groth3, heike köhler1,
katrin Reichwald3, Matthias Platzer3, Manja Marz2
1
institute of Molecular Pathogenesis, Nrl for
Paratuberculosis, Friedrich-Loeffler-Institut
2
rna bioinformatics and High throughput
Analysis, Faculty of Mathematics and computer
science, Friedrich-schiller-university
3
leibnitz institute for Age research, Fritz
lipmann institute
4
Department of Genome Analysis, Helmholtz
centre for infection research
Mycobacterium avium subsp. paratuberculosis
(MAP) - the etiologic agent of paratuberculosis affects cattle, sheep and other ruminants. MAP was
detected in environmental samples, in raw milk and
isolated from man. the etiologic role of MAP in human
Crohn’s disease is under discussion. Deciphering of
phenotypic differences including virulence and host
association observed between cattle and sheep strains
(belonging to MAP-C and MAP-S) by comparative
genome analysis needs data of isolates originating
from different geographic regions of the world.
62
MAP sheep strain jIII-386 (MAP-S) from a migrating
herd in Germany was subjected to whole-genome
shotgun sequencing, de novo assembled, and
annotated by BacProt. NcRNAs were annotated by
homology search of Rfam families using the GORAP
pipeline. Additionally, a newly finished sequence
of cattle isolate jII-1961 from Germany, published
MAP-C strains k10, MAP4 (both from u.S.), MAP-S
draft genomes of strain S397 (from u.S.) and strain
CLIj361 (from Australia), as well as M. a. subsp.
hominissuis strain MAH104 were used for comparison
and assembly improvement of JIII-386. All genomes
were annotated by BacProt and results compared
with NCBI annotation.
With jIII-386, the so far best assembled type-I/III
strain is presented here. Both approaches detected
corresponding protein-coding sequences (CDSs), but
also CDSs that were exclusively determined either by
NCBI or BacProt. A new Shine-Dalgarno sequence
motif was extracted, possibly conserved among
Mycobacteria. Novel CDSs and about 80 ncRNAs
and riboswitches were unveiled for MAP; numbers of
ASpks, G1, ykkC-III differ among MAP-S and MAP-C.
A high sequence conservation of all newly identified
ncRNAs was observed. One or two CDSs encoding
proteins of the PE-PGRS family were identified in MAP
as well as in MAH104. Some previously described
differences between genomes of MAP-S and C could
be partially revised. Four out of ten putative MAP-S
specific large sequence polymorphisms (LSPSs) are
also present in MAP-C strains. Independent of regional
origin, results of protein coding gene annotation and
single nucleotide variant (SNv) analysis confirm the
strong similarity of MAP-C strains, and show higher
diversity among MAP-S strains.
Sequences of two additional MAP strains and the
use of two annotation programs unveiled new
insights in MAP-type specific gene regions and will
help to decipher genes responsible for different host
association and virulence of MAP-S and MAP-C.
P96
IN VITRO ACTIVITIES OF BEDAquILINE
AGAINST NON-TuBERCuLOuS MYCOBACTERIA
Diana Angelica Aguilar Ayala1, jorge Gonzalez
y Merchand2, koen Andries3, Peter Vandamme1,
juan Carlos Palomino1, Anandi Martin1
1
university, Gent
2
Departamento de Microbiologia, escuela
Nacional de ciencias biologicas, instituto
Politecnico Nacional, Mexico
3
Janssen infectious Diseases, beerse
Background: Infections caused by NtM have
become more frequent in the last years since NtM
have emerged as important pathogens in immunecompromised patients. unfortunately, their treatment
is long, toxic and often with poor results. Bedaquiline
(tMC207) has been recently approved for the
treatment of multidrug-resistant tuberculosis and
based on in vitro data showing promising activity
against NtM, there is increasing interest in its
potential use for treating NtM infections.
Objective: The aim of this study was to determine
the Minimum Inhibitory Concentration (MIC) and
the Minimum Bactericidal Concentration (MBC) of
bedaquiline against 11 reference strains of rapid
growing NtM (M. smegmatis, M. fortuitum, M.
chelonae, M. phlei, M. flavescens, M. duvalii, M.
cosmeticum, M. chitae, M. goodie, M. kumamotonense,
M. mucogenicum).
Methods: the MIC was determined by the broth
dilution method using the resazurin microtiter assay
(REMA) and the standard broth turbidity method
(Muller Hinton). the MBC was determined by
conventional plate agar dilution method.
Results: In both assays, REMA and the Muller
Hinton method, range concentration of bedaquiline
was assessed from 0.003 to 2 µg/mL. the MIC of
bedaquiline for 9 NtM ranged between 0.007 - 0.062
µg/ml. Additionally one strain had a MIC of 2 µg/ml
and another a MIC > 2 µg/ml. the MBC for 9 NtM
was found to be higher than 2 µg/ml for the majority of
the strain tested. However, for M. fortuitum the MBC
was 0.062 µg/ml and for M. cosmeticum the MBC
was 0.015 µg/ml.
Conclusion: Bedaquiline exhibited a strong
inhibitory effect against the majority of the rapid
growing NtM species tested (9/11) having MICs
of ≤0.062 μg/ml. However, the MBC of bedaquiline
against the majority of the species was found to be
much higher than its MIC (> 2 µg/ml). An interesting
finding was that 2 NtM species (M. flavescens
and M. duvalii) had significantly higher MICs that
might be considered naturally resistant to bedaquiline
and are under investigation. These results warrant
further investigation to assess the potential use of
bedaquiline in treatment regimens customized for
NtM infections.
P120
IDENTIFICATION OF NON-TuBERCuLOuS
MYCOBACTERIA CLINICAL ISOLATES BY
TANDEM DNA SEquENCING AND MALDI-TOF
MS PROTEIN ANALYSIS
johana Monteserin1, Roxana Paul1, Margo
Cnockaert2, Maria Belén Orandoni1, Beatriz
Lopez1, Palomino juan Carlos2, Peter
Vandamme2, Viviana Ritacco1, Anandi Martin2
1
instituto Nacional de enfermedades infecciosas
Anlis “carlos G. Malbrán”, buenos Aires,
Argentina
2
Background: In our medium-resource reference
laboratory in Argentina, the identification of non
tuberculous mycobacteria (NtM) to the species
level has relied for many years on phenotypic tests
and PRA-hsp65 analysis. Out of about 130 clinical
isolates identified as NtM each year, 25-30 do not
attain definitive species assignation.
Objective: To compare the DNA based methods
using tandem sequencing of four gene loci and the
recently implemented protein analysis using matrixassisted laser desorption ionization-time of flight
mass spectrometry (MALDI-toF MS) for accurate
species identification of NtM clinical isolates that are
difficult to differentiate using our laboratory diagnostic
algorithm.
Methods: We investigated a selection of 40 NtM
clinical isolates obtained in 2002-2013 in Argentina,
which were not identified to the species level and/
or represented iteratively ambiguous phenotypic/
PRA patterns. We performed 16SrRNA, hsp65,
rpob and soda sequencing using an ABI Prism
3130 xL Genetic Analyzer. Analysis was performed
with the Bionumerics software and BLAST in the
NCBI database. MALDI-toF MS was performed
according to the manufacturer’s protocol (Biotyper
System, Bruker Daltonics) for protein extraction from
Löwenstein–jensen slants and spectra were analyzed
against the manufacturer’s database.
Results: tandem DNA sequence analysis allowed
consensus identification of 31/40 (77.5%) isolates
to the species level. Sequencing failed in univocally
assigning species to four isolates of the Mycobacterium
terrae Complex, one of the Mycobacterium avium
Complex, and four unclassified rapid growers. In
pilot MALDI-toF MS protein analysis experiments,
species identification was fully congruous with
DNA sequencing for 18 out of the 20 isolates so far
analyzed.
Comments: Preliminary results show that tandem
DNA sequencing analysis of the selected gene loci
is useful to resolve species assignation of most
difficult to identify NtM clinical isolates in our setting.
Also, MALDI-toF MS protein analysis shows
promise as a tool to identify clinically relevant NtM
but needs further examination and the construction
of customized NtM databases to maximize its
accuracy.
P121
MASTITIS IN COWS CAuSED BY
MYCOBACTERIuM FORTuITuM – A NEW
MOMENT IN DIAGNOSTICS AND TREATMENT
Silvio Spicic1, Miroslav Benić Benić1, Gordan
kompes1, Vera Katalinić-Janković2, Maja ZdelarTuk1, Irena Reil1, Sanja Duvnjak1, Mateja Pate1,
Luka Cvetnić1, Željko Cvetnić1
1
croatian Veterinary institute, Zagreb
2
croatian National institute of Public Health,
Zagreb
63
Mycobacterium (M.) fortuitum is recognized as the
etiological agent of mastitis in cattle and is considered
to be species the most commonly associated with
mastitis among nontuberculous mycobacteria (NtM).
The bacterium has also been isolated from raw-milk
samples collected from bulk tanks and from milk
samples from tuberculin test positive cows.
During February and March 2015, bacteriological
investigation targeting the causative agents of
mastitis was carried out on samples from two older
pregnant Simmental cows from two different herds.
In both cases, the anamnestic data coincided: the
mastitis was persistent and resistant to treatment with
antimicrobials. In one case, the therapy was repeated
several times using antimicrobials of different groups.
Scarce clinical signs included positive reaction to
mastitis test. The milk samples were inoculated onto
esculin-blood agar plate. In both cases, the culture
growth dynamics was identical: the first tiny dust-like
colonies became visible after 48 hours of incubation
at 37°C and reached a diameter of 1-2 mm after 72
hours. Gram-stained smear revealed blue bacilli
while ziehl-Neelsen staining showed the presence
of acid fast bacilli. The isolates were investigated
using conventional PCR, Genotype Mycobacterium
CM (Hain Lifescience) kit and biochemical tests. the
latter identified both isolates as M. fortuitum while the
GenoType assay results corresponded to M. fortuitum
ii/M. mageritense hybridization pattern.
the finding of udder infection with a rapidly growing
NtM species shows an increasing importance of
mycobacteria in milk production pathology. Each case
of udder infections with the evidence of long-lasting
treatment should be suspicious to NtM involvement,
especially because of high antimicrobial resistance.
P174
DECIPhERING ThE VIRuLENCE FACTORS
OF ThE OPPORTuNISTIC PAThOGEN
MYCOBACTERIuM COLOMBIENSE
Monica Natalia Gonzalez Perez1, Martha I
Murcia2, Carlos Parra-Lopez2, jochen Blom 3,
Andreas Tauch 4
1
Microbiology Department, school of Medicine,
National university of colombia, bogotá,
colombia; Medical Microbiology and Genomics,
institute for Genome research and systems
biology, center for biotechnology (cebitec),
bielefeld university, Germany
2
Microbiology Department, school of Medicine,
National university of colombia, bogotá,
colombia
3
bioinformatics and systems biology, Justus
liebig university Giessen, Germany
4
Medical Microbiology and Genomics, institute
for Genome research and systems biology,
center for biotechnology (cebitec), bielefeld
university, Germany
64
The genus Mycobacterium was identified over a
century ago and comprises over 160 species that
are ubiquitous in nature and include free-living
saprophytes, opportunistic pathogens and pathogens
of humans and animals. M. tuberculosis (Mtb) is
considered one of the most dangerous human
pathogens, however, there are a large number of
species known as atypical or Non-Tuberculous
Mycobacteria (NtM) which include up to 150
species. Although they are not considered a public
health problem, their importance is increasing due to
the frequent association with immunosuppression,
especially in HIv/AIDS patients, which is highly fatal.
Mycobacterium avium complex (MAC) contains
clinically important NtM worldwide and is the second
largest medical complex in the Mycobacterium genus
after the Mtb complex. MAC is comprised of several
species which are very close phylogeneticallyrelated, but they are very diverse regarding their host
preference, course of disease, virulence and immune
response. the reasons why these MAC members
vary in diversity are not yet clear and the biology of
pathogenic mycobacteria remains an enigma, despite
their importance in human and veterinary medicine. In
this proposal, we are attempting to find immunological
and virulence-related insights in the M. colombiense
genome as a model of an opportunistic pathogen in
the MAC.
For this study, the M. colombiense CECT 3035
genome was annotated using the GenDB platform.
then, we used different bioinformatics tools to
search for CRISPR systems (CRISPR Finder tool),
prophage sequences (Phast tool), genomic islands
(Island viewer and GIPSy tools),singletons (EDGAR),
protein families (PathogenFinder),and mycobacterial
virulence factors (vFDataBase).We did not find either
CRISPR structures or prophages sequences in the
M. colombiense genome. However, we detected a
total of 45 islands, which contain a total of 420 genes,
70% of those codified for unknown proteins, and 34%
are singletons. We detected a total of 452 singletons,
which represent 8.4% of the M. colombiense genome.
the majority of those (85%) contain Sec signal
peptides and 22% are transmembrane proteins.
We also detected one singleton with LPxtG motif
that is indication of a cell wall localization. Finally,
we detected the M. colombiense virulence factors
that are involve in survival mechanisms inside the
macrophage.
We hypothesize that the presence of these
singletons and virulence-associated genes in the
M. colombiense CECT 3035 genome can serve as
diagnostic indicators for opportunistic pathogenicity.
Furthermore, they contribute to our understanding of
the mechanisms used by pathogenic mycobacteria to
cause disease, which can provide new insights into
designing anti-mycobacterial intervention strategies.
Additional comparative genomic and experimental
analyses are necessary to extend this hypothesis.
P189
DETERMINATION OF DISTRIBuTION
AND ANTIBIOTIC SuSCEPTIBILITY OF
NONTuBERCuLOuS MYCOBACTERIA (NTM)
SPECIES IN CLINICAL SAMPLES FROM ThE
CukuROVA REGION, TuRkIYE
Togrul Nagiyev1, Farhad kohansal1, Emel Yarar1,
Begum kayar2, Taylan Bozok1, Gulfer Yakici1,
Fatih koksal1
1
Department of Medical Microbiology, cukurova
university, Adana, turkey
2
the known species in the population have been also
monitored.
key Words: Agar proportion susceptibility
(APS) method, DNA sequence analysis, hsp65,
nontuberculous mycobacteria (NtM).
Nontuberculous
mycobacteria
(NtM)
cause
serious infections around the world, especially in
immunosuppressed patients by direct transmission
from the environment or after colonization. Difficulties
in identification of these species from Mycobacterium
tuberculosis complex may lead false diagnosis and
treatments. In recent years, increased incidence
of immunosuppressing diseases as Acquired
Immunodeficiency Syndrome (AIDS) have further
increased the prevalence and importance of
nontuberculous mycobacteria (NtM) infections.
More than 140 NtM species have been reported, and
at least 25 of them have been strongly associated
with NtM diseases. the culturing and phenotypic
identification of these bacteria at the species level
is complex and time consuming. And reasons such
as great similarity of the genetic structure and
identification of novel species and subspecies highly
limit molecular diagnosis as well. The hsp65 gene,
which is present in all mycobacteria, is more variable
than the 16S rRNa gene sequence and is therefore
potentially useful for the identification of genetically
related species.
In the study, we aimed to determine distribution
and antibiotic susceptibility of tDM species in the
Cukurova region, turkiye.
In this study, 96 NtM strains isolated from 21918
clinical materials of patients that have pulmoner
tuberculosis and/or suspected contact from Adana
and the neighboring provinces have been identified
to species level by DNA sequence analysis method
targeting the specific hsp65 gene region in the Cu
TDRAC Regional Laboratory for Tuberculosis from
November 2012 to September 2014. Antibiotic
resistance paterns have been evaluated by
Lowenstein Jensen - Agar Proportion tests.
It was observed that the most prevalent NtM isolate
was M. abscessus with ratio of 24,7% (23/93), and
antimicrobial resistance were frequently emerged
against Doxycycline and Moxifloxacin with same ratio
of 17,2% (16/93), whereas lowest resistance rate was
detected against Clarithromycin as % 5,4 (5/93).
In conclusion, considering the great number of
the examined samples, besides determining the
distribution and antibiotic susceptibility of NtM species
in the Çukurova region, turkiye, the movement of
65
GLOBAL PROSPECTIVE OF TB DIAGNOSTICS
P16
P32
COMPARISON OF GENExPERT MTB ASSAY AND
CONVENTIONAL CuLTuRE METhODS FOR ThE
DIAGNOSIS OF TuBERCuLOSIS
Ilhan Afsar1, Aslı Gamze Sener1, Selcuk kaya1
1
Objectives: Diagnosis of tuberculosis (tB) can be
done in many ways. the Genexpert MtB/RIF test
(Cepheid) is a closed-cartridge-based system that
is easy to operate by minimally trained staff and
gives results in approximately 2 h, for TB some of
the century-old tests are still considered as a gold
standard. The aim of this study was to determine
sensitivity and specivity of the Genexpert MtB/RIF
assay in the diagnosis of TB.
Methods: In this study, respiratory and non
respiratory samples studied during the clinical routine
by Genexpert MtB/RIF method and conventional
culture method in IkCu Ataturk training and
research hospital laboratory of microbiology, Izmir,
turkey, between january 2014 to March 2015 were
investigated. Our conventional culture methods are
Lowenstein-jensen culture tube method and MGIt
960 liquid medium system.
Results: A total of 627 clinical specimens included
in the study. of the 627 clinical specimens studied,
17 culture positive and 16 Genexpert MtB/RIF
positive for M. tuberculosis. Four PCR negative
specimens have detected while 17 culture positive
and 3 culture negative specimens have detected
while 16 PCR positive . Results were shown at the
table. the sensitivity and specificity were 76.5% and
99.5%, respectively, (p<0.001). there is no difference
respiratory and non-respiratory samples.
Conclusions: Genexpert MtB/RIF can be used in
laboratories as a fast and reliable test. Also Genexpert
MtB can be recommended safely in non-respiratory
samples.
Table. Positive and negative results of culture and
GenExpert MTb/RiF are shown
Culture
(+)
Culture
(-)
66
GenExpert MtB/
RIF (+)
GenExpert MtB/
RIF (-)
13
4
3
610
EVALuATION OF SPEED-OLIGO DIRECT TB
CASSETTE FOR DIRECT DETECTION OF M.
TuBERCuLOSIS COMPLEx FROM SPuTuM
SPECIMENS
Abiy Aklilu1, Abenezer Feleke1, Chala Chaburte1,
Boja Dufera1, Silvia Blanco1, Pablo Mendoza1
1
Addis Ababa university
Objective: to determine the sensitivity and specificity
of the new Speed oligo Direct tB Cassette, vicell,
Spain (So-DMt)SPEED-oLIGo® Direct tB for the
detection of M. tuberculosis on clinical samples,
comparing the results with: smear examination,
culture and Genexpert MtB/RIF(Cepheid) in Addis
Ababa, Ethiopia.
Materials and methods: A total of 145 sputum
samples were included in the evaluation of the SODMt. one sample per patient was decontaminated
following NALC-NaOH method by Kubica. After
decontamination, ziehl-Neelsen and culture were
performed, and an aliquot was used for the evaluation.
One hundred nine of the specimens were also tested
by xpert MtB/RIF assay. the aliquot was stored at
-20ºC.
Results: taking culture as reference, the sensitivity
and specificity of the So-DMt assay for the detection
of MtBC were 96%, 97.8%, respectively. After
resolution of discrepant results with culture and clinical
data, the corresponding values were 96% (100% in
smear-positives and 89.5% in smear-negatives) and
100%. the concordance between So-DMt assay
results and culture was k: 0.92 (SE 0.0348), indicating
excellent concordance. The sensitivity of both SODMt and xpert MtB/RIF assays was 94.8% (100% in
smear-positives and 87.1% in smear-negatives). the
specificity of So-DMt and xpert MtB/RIF assays
were 100% and 93.8% (100% in smear-positives and
93% in smear-negatives), respectively.
Conclusions:
1 So-DMt has a good sensitivity and specificity in
smear-positive and smearnegative specimens. It’s
use can avoid the need to wait for culture results.
2 So-DMt assay is simple, rapid and provides a
good sensitivity for detection of the M. tuberculosis
complex from decontaminated sputum samples.
3 This assay might be a good alternative to real-time
PCR assays for laboratories not equipped with
real-time PCR instruments.
P65
COMPARISON OF RAPID MGIT TBC
IDENTIFICATION TEST AND MYCOLIC ACID
ANALYSIS BY hIGh PERFORMANCE LIquID
ChROMATOGRAPhY FOR CONFIRMATION OF
Ilhan Afsar1, Yusuf Saglam1, Erkan Yula1, Aslı
Gamze Sener1, huseyin Baskin2, Selcuk kaya1
1
2
school of Medicine, Dokuz eylul university
Aim: A culture confirmation test for the detection of
Mycobacterium tuberculosis complex strains that
uses a lateral-flow immunochromatographic assay to
detect the MPB64 antigen, the MGIt tBc identification
(tBc ID) test, has been developed. We evaluated
the performance of 66 positive TBc ID test of the M.
tuberculosis complex against mycolic acid analysis
by Mycolic Acid Analysis by High-Performance
Liquid Chromatography (HLPC) for identification
of Mycobacterium complex. We compared these
positive 66 TBc ID results to HLPC .
Material and Method: Between January 2010 and
june 2014, clinical samples from IkCu Ataturk training
and Research Hospital were processed using the
MGIt 960 system (BD Diagnostics, Sparks, MD) with
standard protocols. Positive cultures were examined
for acid-fast bacilli (AFB), and AFB-positive cultures
were used to perform TBcID and HLPC analysis
by using the Sherlock mycobacterial identification
system according to manufacturers’ instructions.
Results: During four years 66 clinical specimens
were found positive by TBc ID test. Of the 66 TBc
ID positive test, 63 of them were found positive
by HLPC. In two of the three species identified M.
mucogenicum and one type were determined as M.
abscessus/chelonae by HLPC while identified as M.
Tuberculosis complex by TBcID test. Sensitivity and
specificity of the tBc ID test was found to be 95.5%
and 100%, respectively
Conclusion: the tBc ID test is an easy, very
sensitive, and specific diagnostic tool. Considering
that the BD MGIt tBc ID test is rapid, less laborintensive, and inexpensive, it could be useful test for
in TB diagnostic laboratories.
P66
RELATIONAL SEquENCING TB DATA
PLATFORM (ReSeqTB): GLOBAL
COLLABORATIVE EFFORT TO BuILD A
CENTRALIZED RELATIONAL DATABASE
FOR INVESTIGATING ThE CORRELATIONS
BETWEEN M. TuBERCuLOSIS GENOTYPES
AND DRuG RESISTANCE
Paolo Miotto1, David Dolinger2, Timothy Rodwell2,
Angela Starks3, Daniel johnson4, Marco Schito5,
Matteo Zignol6, Stefan Niemann7, Daniela M.
Cirillo1
2
Foundation for innovative New Diagnostics
(Find)
3
Division of tuberculosis elimination, National
center for Hiv/Aids, Viral Hepatitis, std, and tb
Prevention, centers for Disease control and
Prevention
4
Division of Aids, National institute of Allergy
and infectious Diseases, National institutes of
Health
5
critical Path institute
6
Global tb Programme, tb Monitoring and
evaluation, World Health organization
7
borstel, Germany
1
Rapid drug susceptibility tests (DSts) are needed
to improve the management of tuberculosis (tB)
patients, the rational use of the new tB drug
regimens, and to better understand the global burden
of drug-resistant TB. Having relevant genotypic and
phenotypic information on M. tuberculosis (MtB)
validated and made accessible in one place would
support the development of new tests that can
rapidly inform appropriate therapy for TB patients. A
comprehensive, curated and easy-to-use database
(DB) providing a one-stop data source of this scope
does not currently exist and is a major barrier to the
understanding of the relationship between genetic
mutations and drug resistance (DR) in MtB.
A global partnership of academic institutions, public
health agencies, and non-governmental organizations
has been established to develop a Relational
Sequencing tB Data Platform (ReSeqtB), aimed at
understanding the genetic basis of phenotypic DR by
correlating mutation data with phenotypic DST and
associated patient outcomes ensuring quality and
security of information, high legal and intellectual
property standards, and patient data privacy.
A systematic review of literature for MtB datasets
has been carried out to identify: (i) DBs with DRassociated mutations, (ii) surveillance datasets, (iii)
next generation sequencing (NGS) datasets, and (iv)
genetic variation datasets. The review highlighted
the existence of large collections of genotypic data
(ranging from targeted to whole-genome sequencing)
for more than 50,000 clinical MtB isolates across
70 countries worldwide; >60% of isolates having
associated phenotypic DSt data (primarily for firstand/or second-line drugs). the analysis also revealed
clinical data for more than 30,000 tB patients (mainly
tB treatment history and/or clinical outcomes). these
data are distributed across 368 studies (including 50
DB platforms and 18 web-tools/software).
owners of these data identified through the literature
search will be asked to contribute the datasets for
initially populating the ReSeqtB DB. the inclusion of
these and future NGS data in the ReSeqtB platform
will support development of new diagnostics, provide a
67
validated and updated list of DR-associated mutations,
facilitate clinical decision making, and improve global
surveillance of tB. the ReSeqtB initiative offers an
opportunity for a value-added global collaboration to
achieve improved patient outcomes and to advance
the efforts to control and ultimately eliminate TB.
P79
COMPARISON OF TWO NEW TRANSCRIPTIONREVERSE TRANSCRIPTION CONCERTED
REACTION METhODS (TRCR-160 M.TB and
TRCR-80) FOR RAPID DETECTION OF M.
TuBERCuLOSIS COMPLEx IN RESPIRATORY
SPECIMENS
Antonio Mazzarelli1, Maria Grazia Paglia1,
Alessandro Piscini1, Ornella Butera1, Eugenio
Bordi1, Silvia D’Arezzo1, Antonella Vulcano1,
Carolina Venditti1, Carla Nisii1, Antonino Di Caro1
1
laboratory of Microbiology and bio-repository,
National institute for infectious Diseases lazzaro
spallanzani, rome, italy
Introduction: The rapid and accurate diagnosis
of M.tuberculosis complex (MtBC) infections are
important for the optimal treatment and prevention
of TB infections. Although culture remains the gold
standard for the detection of tuberculosis infection,
various molecular-based methods have been
developed to speed up the diagnostic process.
We have compared two transcription–Reverse
transcription Concerted Reaction systems (tRCR) for
their ability to rapidly detect M.tuberculosis complex
in respiratory samples.
Methods: 201 specimens were collected from patients
treated at L.Spallanzani Hospital in Rome. Samples
were analysed by two TRCR systems (TRCRapid-160
M.tB and tRCReady-80 M.tB, tosoh Bioscience,
tokyo, japan), that use an oxazole yellow-linked
DNA probe and isothermal RNA amplification to
detect 16S rRNA. the tRCRapid-160 M.tB system
is semi-automated and has been used in our routine
activity, while the new tRCReady-80 offers a total
automation of extraction, amplification and detection
analysis. Acid-Fast bacilli (AFB) staining, solid
and liquid medium culture were performed on all
samples. Positive cultures were analysed using an
“in-house” IS6110 MtBC nested-PCR, to confirm the
identification of M.tuberculosis. For tRCRapid-160,
the Extragen M.tB extraction kit was used, while the
TRCReady-80 is an automated system employing
a cartridge-type set of reagents for purification
amplification and detection.
Results: Identification results obtained by tRCR,
AFB staining, culture and PCR were compared to
highlight discrepancies and evaluate Sensitivity
and Specificity of the two tRCR systems. of the
201 specimens, 5 specimens were excluded due
to instrument errors and 4 because they were nonrespiratory specimens; 192 respiratory samples were
68
analysed. The comparison between the two methods
shows that the sensitivity and specificity were 87.2%
and 98.6 respectively. TRCR methods and culture
showed a sensitivity and specificity of 93.2% and
98.6%, 100% and 98% respectively when compared
to identification results. When compared to clinical
data the sensitivity and specificity values for tRCR
were 93.2% and 98.6% for tRCReady-80 and 100%
and 98% for tRCRapid-160.
Conclusions: TRCReady-80 system offers the
advantages of higher automation, in terms of
simplicity of use, speed and safety. Further studies
are needed to confirm these data. the tRCR method
proves clinically useful for rapid identification of
M.tuberculosis complex in respiratory specimens.
P118
INCREASING MYCOBACTERIAL GROWTh
uSING Tika SuPPLEMENTS
Tulika Munshi1, kai hilpert1, Tim Bull1
1
institute of infection and immunity, st George’s
university of london
Introduction: Isolation of mycobacteria is a basic,
gold standard diagnostic technique. unfortunately
extreme slow division rates and tendencies to enter
dormancy by slow growing pathogens such as M.
tuberculosis have proven either restrictively long or
in many cases not possible at all. This is particularly
acute in samples with a low infectious load. New
methods are called for to recover and culture slow
growing mycobacterial species more efficiently.
Methods: using comparative growth curves for M.
tuberculosis H37Rv and other mycobacteria with
various conventional media, we performed a series
of optimisations to develop novel natural based
mycobacteria-specific media supplements (tika) that
when added to Middlebrooks base (solid and liquid)
could significantly improve mycobacterial growth.
Results: We demonstrate that particular TiKa
supplement formulations were able to stimulate M.
tuberculosis and M. avium species growing in both
conventional solid and automated liquid formats. this
included significant decreases in lag phase, improved
division rates in exponential phase and up to 2 log
improvements in recovery from low load serial dilutions
when compared with conventional media alone.
Conclusion: TiKa supplements applied to low
concentration mycobacterial inocula are able to
suspend conversion toward a dormant non-proliferative
phenotype and under selected conditions induce up
to double the division rate of M. tuberculosis and other
mycobacterial strains relative to conventional media.
tika supplements could deliver significant impact
on the sensitivity and rapidity of culture diagnosis in
mycobacterial diseases.
P126
P135
OuR FIRST RESuLTS WITh ThE GENExPERT
MTB/RIF SYSTEM®
Maria Müllerová1
1
Department of Mycobacteriology, citylab Prague
ltd., czech republic
DETECTION OF LATENT TuBERCuLOSIS IN hIVINFECTED PATIENTS FROM SIMON BOLIVAR
AND SANTA CLARA hOSPITALS AT BOGOTá,
COLOMBIA
Martha I Murcia1, Francy johanna Pérez Llanos2,
Magda Beltrán2, Liliana Sánchez2, Carlos ParraLópez1, Myriam Navarrete1, Angélica knudson1,
Ricardo Sánchez3
1
2
3
Departamento de Psiquiatría, Facultad de
Medicina, universidad Nacional de colombia
the great demand for rapid, reliable and price-friendly
laboratory examination of specimens for M.tB, using
molecular biologic techniques and enabling prompt
diagnosis and therapy, is met by the Genexpert
System®. From one sample we have detection
of Mycobacterium tuberculosis and resistance or
susceptibility to Rifampicin (RIF). the test takes only
2 hours. this system is a closed, self-sufficient, fully
integrated, automated molecular biologic platform.
It represents a fundamental change in completely
automated analysis. therefore this unique technique
was introduced in our laboratory, one of the first in the
Czech Republic.
In the Czech Republic there is a low incidence
of tB (2013 – 502 cases, incidence 4.8/100,000
inhabitants; 2014 – 512 cases, incidence 4.9/100,000
inhabitants). However, due to increasing migration of
subjects suffering MDR-tB this favorable situation is
in danger at present.
Laboratory results of subjects examined due to clinical
signs (cough, fever, weight loss), as contacts, or for
suspected TB are presented.
In total 134 samples were examined, from which 4
were positive (3%) and from them 1 was resistant
to RIF, supposition MDR-tB (0.75%), immediate
treatment started, over a period of 6 months.
Extraction, concentration of nucleic acid, purification,
amplification and identification of target nucleic acid is
possible directly from untreated primary specimens.
the laboratory procedure is very simple requiring a
minimum of manual steps. The system generates
exact results in a very short space of time with
minimal risk of contamination. Once the specimen is
deposited in the detection cassette, there is no risk of
contamination or cross-contamination.
The test is irreplaceable in statim examination. It lasts
only 2 hours – it is the fastest of molecular biologic
methods currently in use, at the same time the result
on susceptibility to RIF is obtained.
• The test should always be implemented in suspect
TB.
• The test should always be implemented at start of
therapy.
• The test should always be implemented 2 months
after termination of therapy.
• The test should be performed before start of
biological treatment.
• the test should be performed in quantiFERoN®
TB Gold In-Tube positive patients.
Introduction: One-third of the people around world
have Latent tuberculosis Infection (LtBI). the
lifetime risk of progression to active tuberculosis
for a person with LtBI is estimated to be 5–10%. In
patients co-infected with M. tuberculosis and human
immunodeficiency virus (HIv) the risk is considerably
higher for progression to active tuberculosis (10%
annually). the tuberculin skin test (tSt) is the classic
most used test for the detection of LTBI infection
in Colombia and no evidence base exists for the
selection of interferon-γ release assays (IGRA)
methodology in HIv-infected patients. the aim of this
study is to detect LtBI in HIv patients from two public
hospitals in Bogota, Colombia.
Methods: This is a comparative study that evaluates
the performance of the tSt and quantiFERoN®-tB
Gold In-tube (qF-G-It) for the diagnosis of LtBI in
HIv-Infected patients. From october 2014 to october
2015 we expect to enroll 350 HIv-positive adult patients
(over 18 years old). A total of 72 HIv patients having
all inclusion criteria have been enrolled between
November 2014 and March 2015 at Simon Bolivar
and Santa Clara Hospitals in Bogota, Colombia,
South America. Blood samples were taken and tested
using qF-G-It and tSt (read 48-72 h after). the HIv
/AIDS laboratory central of Simon Bolivar and Santa
Clara hospitals provided information about counts of
CD4+ t lymphocytes detected by flow cytometry.
Results: 75 qF-G-It and tSt were performed.
Among the exposed patients the median CD4 T
lymphocytes count was 136 cells/µL. qF-G-It was
positive in 7 patients (9.3%), negative in 20 (26.6%)
and undetermined in 48 (64%). three patients qFGIt positive had <50 CD4+ cells/µL; two between 50
and 199 cells/µL and two between 200 and 500 cells/
µL. Seven qF-G-It negative patients had <50 CD4+
cells/µL, nine between 50 and 199 cells/µL and four
between 200 and 500 cells/µL. Regarding patients
with undetermined qF-G-It, 25 had <50 cells/µL; 14
between 50 and 199 cell/ µL and in nine between
200 and 500 cell/ µL. on the other hand, tSt was
positive in three patients (4%) and negative (0 mm)
in 72 (96%)
Conclusion: using TST for LTBI screening in
69
Colombian individuals with HIv isn’t a good tool in
Colombia. We suggest including IGRA for higher
detection of LtBI, however, the performance of qFtGIT for diagnosis of LTBI is remarkably affected by
cell counts lower to 500 CD4+cells/µL.
P136
MYCOBACTERIAL INFECTIONS IN hIVINFECTED INDIVIDuALS IN BOGOTá,
COLOMBIA
juan Germán Rodríguez1, Martha I Murcia1,
Magda Beltrán2, Francy johanna Pérez Llanos2,
Liliana Sánchez2, Carlos Parra-López1, Myriam
Navarrete1, Angélica knudson1, Ricardo
Sánchez3
1
2
3
Departamento de Psiquiatría, Facultad de
Medicina, universidad Nacional de colombia
Introduction: Nowadays tuberculosis (tB) is the
most important infectious disease worldwide and one
of the major causes of mortality in people living with
acquired immune deficiency syndrome (HIv/AIDS).
In Colombia, with a national incidence of 25 cases
by 100.000 people, in the year 2014 were reported
12.415 cases of tB, 2.029 cases of HIv/tB coinfection and 608 deaths by TB. Mycobacterium avium
complex (MAC) infection is a common complication
of advanced acquired immunodeficiency syndrome
(AIDS) disease and is an independent predictor of
mortality and shortened survival the objective of
this study is to detect mycobacterial infection in HIvinfected patients with clinic suspicious of TB in two
public hospitals in Bogota, Colombia.
Methodology: This is a descriptive cross-sectional
study. Between October 2014 and October 2015
we expect to enroll 350 adults patients HIv-positive
(over 18 years old), with clinical suspicious of tB.
A total of 564 clinical specimens (mostly paired
samples of blood, pulmonary) and extra-pulmonary
samples were collected from 162 HIv positive
patients. The samples were processed for acid-fast
bacilli (AFB) stain, culture and Multiplex polymerase
chain reaction HSP65-IS6110 PCR. Susceptibility
test was determined by baCtEC MGit 460 SIRE®
methodology and Genotype MtBDRplus®
Results: In total 162 patients were enrolled up to
now. 132/162 patients (81%) were men and 30 (19%)
women (ratio male / female of 4.4:1), with an average
age of 38 years. overall 33 of 162 patients (20.4%)
were positive for any Mycobacterium species by acidfast staining or culture. Mycobacterium tuberculosis
complex (MtC) was identified in 24/162 patients
(14%), Mycobacterium avium complex (MAC) was
isolated in 4/162 individuals (2.5%) and two patients
(1.2%) presented simultaneous infection by MtC
and MAC. the AFB smear was positive in 28/240
70
(12%) samples. Among 92 clinical samples tested by
PCR Multiplex six (6.5%) were positive for HSP656110. Drug susceptibility performed in 10 cultures
of MtC demonstrated that one (10%) was multidrug
resistant (MDR). 29 clinical specimens evaluated
by MtBDRplus Genotype ver2.0®, 14 (48%) were
positive for MtC, 13 were sensitive to isoniazid
and rifampicin (93%), while 1 (7%) was sensitive to
isoniazid but resistant to rifampicin.
Conclusion: these findings suggest usefulness of
different methodologies for detecting mycobacterial
infections in HIv-Infected patients in order to establish
strategies for rapid diagnosis and treatment of TB in
this population.
P138
A POST IMPLEMENTATION REVIEW OF ThE
CEPhEID xPERT MTB RIF ASSAY VERSION G4
FOR ThE DIAGNOSIS OF PuLMONARY AND
ExTRA-PuLMONARY TuBERCuLOSIS IN A
LARGE ACuTE TERTIARY CARE INSTITuTE
Azhar hamdan1, justine Woei Ling Peh1, Yen Ee
Tan1, Li-hwei Sng1
1
singapore General Hospital
tuberculosis (tB) is a major health problem worldwide
and its incidence is 36.7 per 100,000 in Singapore.
Local cases of drug-resistant TB have also been
increasing; thus quick and accurate diagnosis is
essential for controlling its spread. An initial validation
study using the Cepheid xpert MtB/RIF Assay
version G3 on 179 pulmonary and extra-pulmonary
specimens had been performed from 2010-2011 at
the Central tuberculosis Laboratory. Subsequently,
the laboratory evaluated and implemented the use
of version G4 in 2012. A review was performed to
assess the post-implementation test performance in
the routine diagnostic laboratory.
All samples received between October 2012 and
December 2014 were included in the study, with
culture being used as the gold standard. Clinical case
reviews were performed for discrepant cases. Of the
569 clinical samples, 546 had evaluable results. the
sensitivity and specificity for pulmonary specimens
was 89.3% (95% Confidence Interval [CI]: 78.5 –
95.0) and 90.5% (CI: 84.6 – 94.2); versus 68% (CI:
54.2 – 79.2) and 95.2% (CI: 92.1 – 97.1) for extrapulmonary specimens. Compared to the earlier
validation study, there was no significant difference
in the test sensitivity (p=0.78 for pulmonary, p=0.64
for extra-pulmonary) and specificity (p=1.0 and
p=0.45). the xpert MtB/RIF was easily implemented
for routine testing with minimal technical expertise
required. However, the overall test performance was
affected by the specimen type, prior antimicrobial
treatment and equipment issues.
P141
DIAGNOSIS OF MYCOBACTERIuM
TuBERCuLOSIS FROM PLEuRAL FLuID IN
BOGOTA, COLOMBIA
Martha I Murcia1, Saray Ossa2, juan Germán
Rodríguez1
1
Grupo Micobac, Departamento de Microbiología,
Facultad de Medicina, universidad Nacional de
colombia
2
Introduction: Pleural tuberculosis is responsible for
30 – 80% of all pleural effusions and may complicate
tuberculosis in 31% of all cases. In Colombia
pleural tB is the first extrapulmonary presentation
in frequency. Currently the diagnosis of pleural
tuberculosis is wasteful, due to the paucibacillary
nature of the samples and the inspecific symptoms of
the patients. Methods such as smear microscopy and
culture in pleural fluid, have a low sensitivity (5% and
40% respectively) and pleural biopsy is an invasive
procedure that can bring problems to the patient; for
this reason the search for new diagnostic techniques
more sensitive and specific is needed.
Methodology: In this study, we examined the
diagnostic efficiency of different techniques in
pleural effusions from patients with clinical signs
and symptoms of pleural tuberculosis. Molecular
methods: IS6110-PCR, RT-PCR iS6110, Genotype
MtBDR plus 2.0, Multiplex PCR (IS6110-hsp65).
Phenotypic methods: ziehl Neelsen staining and
culturing on Löwestein-jensen, Stonebrink medium
and Mycobacterial Growth Indicator tube (MGIt)
incubated in MGIt 460 BACtEC instrument (BD
Diagnostics, Sparks, MD, uSA).
Results: A total of 30/80 pleural fluid were studied.
All samples were lymphocytic exudates, mean 5300
for leukocyte cell/mm3 and lymphocytes of 68%,
with more than 980 Iu / L of LDH value. the smear
microscopy and cultures were negative in all samples,
demonstrating the low sensitivity of these methods in
tuberculous pleurisy. IS-6110 RT-PCR was positive
in 9/30 samples (30%), followed by PCR-IS6110
with a total of 6 positive samples (20%), Genotype
MtBDRplus 2.0 was positive in 5 samples (16.6%),
and the Multiplex PCR (IS6110-hsp65) was positive
in 3 samples (10%).
Conclusion: tuberculous pleural effusions are
mainly exudates with a high burden of cellularity
mainly lymphocytic type. The RT-PCR IS6110 was a
noninvasive, rapid and sensitive tool for the diagnosis
of pleural tuberculosis.
P145
uNDIAGNOSED TuBERCuLOuS
OSTEOMYELITIS OF ThE TIBIA CAuSED BY
MYCOBACTERIuM TuBERCuLOSIS COMPLEx;
A CASE REPORT
Gülnur Tarhan1, Sadık Akgun1, Abdülkadir Sari2,
hakan Sayiner3, Bülent Petik4, Ismail AĞIR2,
H.İbrahim Erdoğdu5, Şükrü Mehmet Ertürk4
1
Faculty of Medicine, Medical Microbiology
Department, Adiyaman university
2
Faculty of Medicine, Department of
Orthopaedics and Traumatology, Adıyaman
university
3
Faculty of Medicine, Department of infectious
Diseases and Clinical Microbiology, Adıyaman
universit
4
Faculty of Medicine, Department of radiology,
Adıyaman University
5
Faculty of Medicine, Department of Patology,
Adıyaman University
Tuberculous osteomyelitis caused by Mycobacterium
tuberculosis complex is very rare event in patients.
the nonspecific nature of the symptoms and specific
diagnosis leads to a delay ranges from a few weeks
to up to several years. the term ‘Brodie’s abscess’
was applied to localized bone abscess that developed
without prior systemic illness. Curettage of the
lesion and the histopathological examination of the
material obtained are necessary for confirmation of
the diagnosis and offer a chance for early healing.
The diagnosis of tuberculosis osteomyelitis is usually
made on clinic and radiological features. But, definitive
diagnosis of tuberculosis can only be made by
microbiological examination from a clinical specimen
taken from the patient. In our study, we report a case
of Brodie’s abscess, a special form of osteomyelitis,
caused by Mycobacterium tuberculosis complex. A
65-year-old man presented at the outpatient clinic with
pain and swelling of leg for the last six months. There
was history of injury for twelve years ago. there was
no tB history (of cough, expectoration, haemoptysis,
swelling elsewhere or other joint involvement.) Past
history of major illnesses like tuberculosis, diabetes
mellitus, repeated blood transfusion suggestive
of congenital haemolytic anaemia, was absent.
The family history was non-contributory. Surgical
evacuation of the pus and curettage were performed.
The curetted tissue was sent for cytological and
histopathological examination and polymerase
chain reaction (PCR) analysis for mycobacterium
tuberculosis. The cytological examination revealed
a granulomatous process and the histopathological
examination found caseating necrosis, epitheloid
cell granulomas, and Langerhans giant cells. the
PCR was negative. At the same time, the specimens
were processed examined by smear microscopy,
conventional solid Löwenstein jensen culture. EzN
staining was negative. Lj cultures were positive in five
weeks. the isolate was identified by using Genotype
71
MtBC and CM/AS assays CM/AS (Hain Lifescience
GmbH, Nehren, Germany) and the isolate was
recognized as MtBC. After identification, the isolate
was tested by the Genotype MtBDR plus assay
(Hain Lifescience GmbH, Nehren, Germany) for drug
resistance. The test was negative for isoniazide and
rifampicin resistance.
P153
“TB-SPRINT” ON SPuTuM DNA ExTRACTS
FROM EThIOPIA: FIRST COMPARISON TO
GENExPERT AND GENOTYPE MTBDRplus
Barbara Molina-Moya1, Silvia Blanco2, Mulualem
Agonafir3, Michel Gomgnimbou4, Lizania
Spinasse4, Guislaine Refrégier4, Daniel Datiko5,
Luis Cuevas6, jose Dominguez1, Christophe
Sola4
1
Germans trias i Pujol.; institut D’investigació
Germans trias i Pujol. universitat Autònoma de
barcelona; ciber enfermedades respiratorias
(ciberes), instituto de salud carlos iii
2
Department of Microbial, cellular and Molecular
biology, college of Natural sciences, Addis
Ababa university
3
infectious and other Diseases research
Department, ethiopian Health and Nutrition
research institute, Addis Ababa
4
orsay; centre Muraz, bobo-Dioulasso, burkina
Faso
5
lstm, university of Hawassa
6
liverpool school of tropical Medicine
tuberculosis (tB) is still one of the most deadly
curable infectious diseases. The aim of this work
was to perform high-throughput spoligotyping and
detection of mutations conferring drug resistance in
Mycobacterium tuberculosis strains circulating among
tB patients in Ethiopia. For this purpose, 56 sputum
specimens were collected, and ziehl-Neelsen (zN)
staining and smear microscopy examination were
performed. DNA was extracted from the specimens
either by a microbeads/lysis method or following
Genotype MtBDRplus (Hain lifesciences, Germany)
manufacturer’s instructions. Genexpert MtB/RIF
(Cepheid, uSA) and/or MtBDRplus were performed
following manufacturer’s instructions. DNA extracts
were analyzed by TB-SPRINT. This method is
based on simultaneous genotyping of the CRISPR
(clustered regularly interspaced short palindromic
repeats) region, rpob, katG, and the promoter region
of inha, by hybridization on microbeads and detection
with a Luminex device (Luminex Corp, Austin, tx).
Forty-eight sputum specimens were positive for
zN staining, four specimens were zN-negative,
and for the remaining four specimens this result
was unknown. of 56 extracted DNA, 55 DNA were
analyzed, and for 49 DNA interpretable spoligotyping
72
results were obtained (genotyping rate = 89.1%). the
global distribution among the 49 interpretable patterns
was observed as follows. Twenty-four patterns were
classified as t3-EtH: a single cluster was found (SIt
149, n=22), and two patterns were orphan. A single
cluster was classified as CAS1-kili (SIt 21, n=8); two
clusters belonged to CAS1-DELHI (SIt 26, n=2; SIt
25, n=2), a single cluster was classified as t1 (SIt
53, n=2); a single cluster was classified as H3 (SIt
699, n=2); and nine orphan patterns were found. of
the 55 DNA samples analyzed, molecular resistance
results by TB-SPRINT were obtained for 40 samples
(72.7%). Sensitivity of tB-SPRINt for detecting
rifampicin resistance considering Genexpert and/
or MtBDRplus as reference was 96.9% (31/32). In
contrast, for the 7 samples susceptible to rifampicin
by Genexpert and/or MtBDRplus, the mutation rpob
531ttG was detected by tB-SPRINt. For isoniazid
resistance, sensitivity and specificity of tB-SPRINt
were 75% (3/4) and 100% (2/2), respectively. these
results, although requiring to be applied at a broader
scale, suggest that tB-SPRINt could be applied
directly on sputum and could help taking early
decisions on the treatment and management of TB
patients.
P162
COMPARATIVE AND COMPuTATIONAL
ANALYSIS OF RuSSIAN M. BOVIS BCG STRAIN
Ruslan Ludannyy1, Maria Alvarez Figueroa1,
Diana Levi2, Natalia Aleksandrova2, Michail
Markelov1, Vladimir Dedkov1, German Shipulin1
1
Moscow, russia
2
Federal State Budgetary Institution “scientific
center for expertise of Medical Application
Products” Ministry of Health of the russian
Federation
The effect of nucleotide replacements might be
more affective on cells’ biogenesis than prediction of
evolution divergence and intrapopulation variability.
The tunnel and ligand mutations or replacements in
active binding sites able to play a more critical role
in cells’ pathway and finally determine a selective
priority by that increasing of bacterial virulence or
pathogenicity. Regardless of permanent or transient
those phenomenon in vivo, for vaccine strains as
example, this modification is actual, where, the
genome stability is a key factor for efficiency and safe
application in prevention medicine.
the molecular genetic technique and global
bioinformatics
analysis
were
applied
for
characterization of genome and categorization of
functional genetic elements M. bovis BCG strain,
which used for vaccination in territory of modern
Russia from 1925 up to date. The NGS technologies
of Roche and Illumina have been performed
independently for sequencing libraries, constructed
by NExtERA and GS FLx kits. the Amos algorithm
for simultaneous assemble (Geneious ver. 7.1.14),
GATK for validation and Glimmer in common with
Blast2go and NCBI database for annotation were
applied. The chromosome of M. bovis BCG Tokyo
172 used for mapping (similarity 96.5%). Additionally,
other strains from Du2 inc. wild strain were aligned
for comparative analysis. In fact, the Russian strain
(grp. 117339) has 7 373 972 bp with G+C contain
of 65.9%. In total, 3750 CDS and 3850 total gene,
3 rRNA, 45 tRNA genes have been annotated.
the 170 repeats, included 36 tE-elements were
predicted by the EDAM ontology database, tRF and
repeat masker. The 182 variability regions have been
detected in the genome, which characterized from
1 to 3 bp mutations. All variability sites have been
verified by Sanger sequencing.
The calculation of evolution rate with Bonferroni
correction has been created for selection of actual
mutations in coding regions, which were formed after
attenuation (1908-1921 yy.). the 3D macromodeling
was performed by ROSETTA with prediction of
functional sites and active center by SABER. For
example, the mutation c. 581 G>C (p. Gly 194 Ala)
was detected in м AtP-synthase subunit β (atpD)
formed around 1960 (±2) year, after calculation.
The mutation effect on secondary structure was
observed, however, the ligand (p. GGAGvGkt 171178) and active binding sites were not affected. the
tunnel simulation analysis by CAvER resulted in
identification three dominant pathways corresponding
to general tunnels in atpD subunit of BCG substrain
(Russia), in contrast of two pathways of wild and other
substrains from Du2 group. kinetics asymmetry in
synthesis process of ATP might be suggested and
requires of further investigation. In conclusion, this
approach for analysis structure modification of coding
regions in common using NGS technology may apply
for evaluation of biological features of mycobacterial
strains in further.
P188
APPLICATION OF IN VIVO MICROBIAL ANTIGEN
DISCOVERY (InMAD) FOR ThE IDENTIFICATION
OF M. TuBERCuLOSIS CIRCuLATING ANTIGENS
IN AN ExPERIMENTAL MuRINE MODEL
Iris Estrada-García1, Bibiana Patricia RuizSánchez2, jessica Castañeda-Casimiro2,
Alejandro Francisco-Cruz3, jeanet Serafín
López4, Isabel Wong-Baeza2, Dulce MataEspinosa3, Rogelio hernández-Pando3, Sergio
Estrada Parra2
1
National school of biological sciences, ipn
2
Depto. de inmunol., encb, ipn
3
Patología experimental, incmn, s. Zubirán, ss
4
escuela Nacional de ciencias biológicas, ipn
In vivo Microbial Antigen Discovery (InMAD) was
described by Nuti et al. (MBio, 2011) as an strategy
to identify novel biomarkers (antigens or antibodies)
as potential targets for diagnosis of two intracellular
bacterial diseases. Tuberculosis is also caused by an
intracellular pathogen that has been spread because
of several factors, including the lack of a rapid
diagnostic test. In this work we used this strategy
to identify circulating antigens in the experimental
tuberculosis mice model described by HernándezPando et al. (CEI, 1996).
InMAD sera: individual serum samples from two
groups of Balb/c mice infected with M. tuberculosis
H37Rv (Mtb) (n=5, days 21 and 60 post-infection),
were mixed with Titermax® and used to immunized
syngeneic mice (i.e. Balb/c). Control sera was
obtained from mice immunized with uninfected mice
sera plus Titermax® or Titermax® alone. Western blot:
three different Mtb antigen cocktails were separated
using a 3/12% SDS/PAGE and reducing conditions:
total extract (MttE), soluble extract (MtSE) and
culture filtrate secreted proteins (CFPS). Antigens
were transferred to PvDF membranes and probed
with InMAD sera days 21 or 60, and control sera.
2nd antibody was goat anti-total mouse Ig´s-HRPo.
Bands were developed with diaminobenzidine and
hidrogen peroxide.
As expected InMAD sera from uninfected mice or
titermax® immunized mice showed no bands. InMAD
immune sera from days 21 and 60 post-infection
showed reactivity with seven and five different Mtb
antigens, respectively. Five protein bands from day
21 were eluted from PvDF membranes and antigens
released using 1%tritonx-100/2% SDS/50mMtrisHCl pH8.8. Samples were separated on a 3/10% SDSPAGE and identified by MS. We considered those
proteins with a match for expected molecular weight
and > 50 % identity. Results showed: Chaperone
protein Dnak 66.7 kDa, Glutamine synthetase 53.6
kDa, Adenosylhomocysteinase 54.3 kDa, Elongation
factor tu 43.5 kDa, Acetyl-CoA acetyltransferase
40.9 kDa and FHA domain-containing protein 16.9
kDa. using the InMAD strategy we identified putative
antigenic targets that may be useful as tuberculosis
biomarkers. Interestingly we did not find other Mtb
antigens previously described as immunodominant,
although Ag85 was detected but only with a 30 %
identity.
We look forward to continue this line of research,
confirming the utility of these antigens using 2D gels
and sera of tuberculosis patients.
P199
EVALuATION OF ThE FLuOROTYPE® MTB
ASSAY FOR uSE WITh NON-DECONTAMINATED
SPECIMENS AT ThE IRISh MYCOBACTERIA
REFERENCE LABORATORY (IMRL)
Philomena Raftery1, Margaret Fitzgibbon1
1
irish Mycobacteria reference laboratory,
labmed Directorate, st James’s Hospital
73
Over the past decade the diagnostics repertoire
for tuberculosis (tB) has expanded significantly,
with several technologies showing promise. In
order to reduce the global burden of tuberculosis
rapid detection methods are required for timely
and effective intervention strategies and effective
treatment of TB cases. Hain Lifescience have
developed the Fluorotype® MtB, a real-time PCR
assay for the direct detection of the Mycobacterium
tuberculosis complex (MtC) from decontaminated
pulmonary and extra-pulmonary specimens. A
previous study (Hoffmann-thiel 2012), performed
in a low-tB prevalence setting, demonstrated an
overall specificity and sensitivity of 98.9% and 88.1%
respectively when compared to culture. The assay
performed well on decontaminated smear-positive
specimens with a sensitivity of 100% which dropped
to 56.3% for smear-negative specimens. An excellent
positive predictive value (100%) in smear-positive
specimens demonstrated its utility in discriminating
between non-tuberculosis mycobacteria (NtM) and
MtC infection.
The aim of the present study was to assess the
performance of the Fluorotype® MtB Assay using
direct specimens (without prior decontamination)
as the starting material. In a prospective study,
specimens from known TB positive patients were
selected, and paired aliquots were collected preand post-decontamination for analysis with the
Fluorotype® MtB Assay. A manual extraction using
the Fluorolyse kit was performed before running
batches of paired samples on the Fluorocycler.
The performance characteristics of the assay were
evaluated for detection of MtC in non-decontaminated
patient specimens and results compared to smear
microscopy and conventional culture, including
clinical information where relevant.
The assay performed well for non-decontaminated
specimens demonstrating a sensitivity of 94.4% and
specificity of 100%. Similar to previous studies, a
high sensitivity value (94%) was achieved on smear
positive specimens. However, the decontamination
protocol (2% NaoH) used at the IMRL appears to
have caused some PCR inhibition as a dramatically
reduced sensitivity (46%) was noted along with
a high proportion of invalid results (30%) on the
decontaminated cohort of samples. This anomaly will
be investigated further in an extension of the study.
From the data presented, the assay can be used
to provide valuable, rapid and clinically relevant
information directly from non-processed specimens.
In a low tB setting, this assay is a useful screening
tool to replace conventional microscopy with the aim
of increasing the sensitivity and speed of diagnosis
of tuberculosis. However, it should not be considered
a substitute but rather an adjunct to traditional
culture methods.
74
P216
ThE EFFICIENCY AND RELIABILITY OF
COMMERCIAL Tk L ANTI TB PNB kIT
FOR RAPID ANTI-TuBERCuLOSIS DRuG
SuSCEPTIBILITY TESTING
Ismail Ceyhan1, Zehra Ortarık1, Resul Altınsoy1
1
Atatürk chest Diseases and chest surgery;
training and research Hospital, Ankara, turkey
tk Media are rapid colorimetric mycobacterial
culture media. the color of tk Media changes from
red to yellow by mycobacterial growth or to green
by contamination. tk Media can be monitored both
visually and in automated incubator reader, Mycolor
TK. TK SLC L medium used for mycobacterial
isolation from clinical specimens and media in TK L
Anti tb PNB kit, used for susceptibility testing, are
ready to use liquid media and they are produced in
plastic tubes since almost one year. this is the first
evaluation of efficiency and reliability of tk L Anti tb
PNB kit since its production in plastic tubes. The kit
was tested by 20 Mycobacterium tuberculosis (M tb)
collection strains obtained from WHo, with known
susceptibility patterns. For this purpose, suspensions
of mycobacteria were prepared and diluted using
the tubes present in the kit and then inoculated to
control and drug containing tubes including the one
with para-nitro-benzoic acid (PNB), for differentiation
of M tb complex bacteria and Mycobacteria other
than tuberculosis (Mott). All strains were identified
to be susceptible to PNB. The susceptibility to INH
and Rifampin of all 20 strains and in 19 strains
for streptomycin and ethambutol, were correctly
identified. While one strain, which was resistant to
streptomycin was misdiagnosed as susceptible, one
other strain, which was susceptible to ethambutol,
was misdiagnosed as resistant.
TK L Anti Tb PNB Kit can be considered a reliable tool
for anti-tuberculosis drug susceptibility testing. Being
ready-to-use, it eliminates contamination and errors
that may occur during preparation of anti-tuberculosis
drug containing tubes in other rapid culture systems.
Extended evaluation studies including larger number
of strains will be valuable.
hOST-PAThOGEN INTERACTION
P23
ThE SIGMA FACTOR SigD OF MYCOBACTERIuM
TuBERCuLOSIS ENhANCES ThE
TRANSCRIPTIONAL ExPRESSION OF ThE
SEPTuM SITE DETERMINING PROTEIN IN
STATIONARY PhASE
Nora Rios-Sarabia1, Miguel Angel Ares2, jorge
Gonzalez-y-Merchand3
1
instituto Mexicano del seguro social
2
unidad de investigación Médica en
enfermedades infecciosas y Parasitarias, centro
Médico Nacional siglo XXi, instituto Mexicano
del seguro social; laboratorio de Microbiología
Molecular, escuela Nacional de ciencias
biológicas, instituto Politécnico Nacional;
escuela Nacional de ciencias biológicas,
instituto Politécnico Nacional, México
3
National school of biological sciences, ipn;
intituto Politecnico Nacional; Dept. of Mirobiol.,
Natl. sch. biol. sci., ipn
The M. tuberculosis genome encodes 13 putative
sigma factors, which controls gene expression in
response to specific extracellular signals. Some
sigma factors regulate housekeeping genes but
others denominated alternative sigma factors have
been identified as regulators responding to specific
environmental stimuli and stress. The alternative
sigma factor SigD of M. tuberculosis controls the
expression of genes whose expression is normally
induced during stationary phase in vitro. the Mt3760
gene of M. tuberculosisCDC1551 codes for the Septum
site determining protein (Ssd), which promotes the
formation of elongated cells by inhibition of septumin
response to the stressful environment. In order to
investigate if SigD could be related to ssd regulation,
we determined the transcriptional expression profile,
performing qRt-PCR in M. tuberculosis CDC1551
wild-type and ∆sigD strains cultured in vitro in
exponential and stationary phases. M. tuberculosis
CDC1551 wild-type and ∆sigD strains were cultured
in Middlebrock 7H9 liquid medium supplemented
with 10% of oADC. total RNA was isolated in the
exponential and stationary phases qRt-PCR was
performed in a LightCycler 480 instrument using
SyBR Green I (Roche), 16S rRNA was used as a
reference gene for normalization. Statistical analysis
for differential gene expression was performed using
the one-way Anova and tukey´s multiple comparison
test. We found that the transcriptional expression of
ssd in the wild-type strain was significantly higher in
stationary phase. Interestingly in the ∆sigD strain the
ssd gene was transcribed at the same level in the
exponential and stationary phases. We conclude that,
these results show the potential of SigD to induce
over expression of ssd gene in stationary phase.
These data could suggest that SigD activates the
transcriptional expression of ssd gene in response to
the stressful environment of stationary growth phase
in vitro.
P38
PhAGOCYTE CELLS BACTERICIDE
ChARACTERISTICS IN PATIENTS WITh
PuLMONARY TuBERCuLOSIS
Marina Dyakova1, Olga Titarenko1, Diljara
Esmedlyaeva1, Viatcheslav Zhuravlev1
1
research institute of Phthisiopulmonology
Background:
resistance
of
Mycobacterium
tuberculosis (Mtb) determined by molecular-genetic
mechanisms depends on intracellular cooperation of
mononuclears (Mn) and neutrophils (N) which is a
basis of the common phagocyte domen functioning.
Peculiarities of coordination of Mn and N activities
and Mtb resistance are thought to be responsible for
results of antituberculosis therapy.
Objective: to study blood Mn and N functional status
in patients with infiltrative pulmonary tuberculosis
(IPt) eliminating drug susceptibility (DS) and resistant
(DR) Mtb.
Methods: Mn and N bactericide functions were
estimated using the following criteria: for oxygenindependent function - levels of lactoferrine (Lf)
and elastase (El), for oxygen-dependent function –
characteristics of nitroblue tetrazolium (NBt) recovery
test - spontaneos (NBts), induced (NBtin) and their
correlation – index (NBtin/NBts), for nitrolizing
function – concentration of nitrite (No). 90 patients
with IPT were divided in two groups: the 1st eliminating
DS Mtb (n=51) and DR Mtb (n=39).
Results: in both groups of patients there were
found similar but quantitavely different changes
of Mn and N characteristics: increase of oxygenindependent activity and decrease of NO generation.
Characteristics of oxidative stress were in referent
range for Mn with a tendency to increase for N. there
was found a difference in structure correlation in
compared groups: only in the 1st (DS) a significant
positive correlation was revealed between indices
of oxidative and nitrolizing types of stress in Mn
and N, synergy changes in oxygen-dependent and
independent functions in both types of cells. Similar
correlations are absent in patients with DR Mtb.
Conclusion: according to the results of the study it
is suggested that Mtb resistance does not influence
the bactericide function of the phagocytes but is
associated with changes in intercellular cooperation.
75
P45
PARTICIPATION OF NON-CODING RNAs IN
ThE ADAPTATION OF MYCOBACTERIuM
TuBERCuLOSIS TO A LIPID ENVIRONMENT
M Ares1, Maria Carmen Menendez2, jG
Rodriguez3, AC hernandez3, j Gonzales-yMerchand4, C helguera4, jR Bustos3, jM Anzola3,
MM Zambrano3, Mj Garcia5, P Del Portillo3
1
instituto Politecnico Nacional México
2
Facultad de Medicina, universidad Autónoma de
Madrid
3
corporación corpogen
4
instituto Politecnico Nacional, México
5
universidad Autónoma de Madrid
*mcm and MA contributed equally to the work
Lipids are a main energy source of M. tuberculosis
during human infection. Our previous study has showed
a set of changes that marks the global transcriptional
adaptation of the tubercle bacilli to a lipid environment
(Rodriguez et al, 2014). Nevertheless the number of
transcripts was lower in the presence of fatty acids
(FA), non-coding RNAs (ncRNAs) are among the
more transcribed products, in particular during the
stationary phase in the FA enriched environment.
In order to have insight into the role putatively played
by those ncRNAs in the lipid environment, we applied
the programme http://cs.wellesley.edu/~btjaden/
TargetRNA2/ to ncRNAs that showed high level of
expression in the conditions studied. That programme
gives information on the target sequences where
the ncRNAs putatively links, along the genome of
M. tuberculosis, as well as the digital hybridization
energy and the significant value of the link detected.
Main features found:
1. three of the five ncRNAs showing highest
expression link to upstream regions of genes
members of the type seven secretion system
(t7SS). Interestingly, the genes showed significant
higher expression in dextrose compared to FA.
Thus probably indicating the attenuation of the
bacteria in the lipid environment.
2. other interesting target was the gene iniB, member
of an efflux pump. this pump could be acting during
stationary phase in FA.
3. A link to acyltransferase, differentially expressed in
FA compared to dextrose, was also detected for
another ncRNA. The gene was found to be essential
in the final step of the penta-acyltrehaloses (PAt)
biosynthesis.
4. A link to a toxin-antitoxin system was also detected
for another ncRNA. This nc could activate the T/A
system in dextrose during stationary phase.
The work demonstrated that knowledge on the
participation of non-coding regions is relevant
for a better understanding of the tuberculosis
pathogenicity.
Acknowledgements:
We thank J Gonzalo-Asensio for his advice on nc-
76
RNA.
Funding resources: International Cooperation CEALuAM and the Spanish Ministry of Health (AES, PI1301218).
P97
ThE GROWTh RATE OF ThE STRAINS
MYCOBACTERIuM TuBERCuLOSIS BEIjING
ISOLATED BEFORE AND DuRING TREATMENT
AND IN ThE TIME OF PASSAGES ON
LOWENSTEIN-jENSEN MEDIuM
Olga Manicheva1, Marine Dogonadze1,
Natalia Melnikova1, Viatcheslav Zhuravlev1,
Anna Zmaznova1, Anna Vyazovaya2, Igor
MOkROuSOV2, Olga Narvskaya3, Boris
Vishnevskiy1
1
2
3
st. Petersburg Pasteur institute; research
institute of Phthisiopulmonology
Background: Growth rate (GR) Mycobacterium
tuberculosis (Mtb) in the environment is determined
by the genetic capacity of strain to adaptation and
depends on the stage of treatment.
Aim: - to estimate the GR on a nutrient medium Mtb
strains with the same spoligotype and similar drug
resistance isolated before and during treatment and
after serial passages.
Methods: We examined GR of 30 MDR or xDR Mtb
strains of Beijing lineage isolated from pulmonary
tB patients, 10 of these strains with the same type
of mutations in genes rpob and katG had passaged
on Lowenstein-jensen medium (10 generations)
and determined the affiliation to the cluster B0/
W148 (Mokrousov et al., 2012). Fluorescence of
Mtb suspension (1 u McFarland deluted 40-fold in
7H9 broth with 10% oADC, 200 µl/well, 30 µl 0.01%
resazurin) was measured daily for 7 days. Growth
curve was constructed by calculating of mean value
of fluorescence, the slope of this curve was calculated
as conventional unit (cu) of GR. GR mean value and
95% confidence interval was calculated: 1) for Mtb
isolates from untreated patients (group I, n=12) and
treated ones (group II, n=18), 2) for isolates after
3rd and 10th rounds of passages, in comparison
with H37Rv. the significance of differences between
groups was determined by t-test.
Results: the Mtb GR was 293.8+42.9 cu in group I,
197.8+68.6 cu - in group II (p=0.001). Relatively strain
H37Rv GR strains (3rd generation) untreated patients
(n=4) accounted for 75.7-100%, untreated with further
treatment failure - 62.4, 65.4 and 68.8%, patients with
a bacterioexcretion history more 2 years - 6.7, 14.3
and 153.5%. A similar pattern was observed at the
10 th generation, except for two strains. 7227 strain
(cluster B0/W148, treatment history of >2 y) was
distinguished by fast growth, its GR was in 1.5 and
1.6 times greater than the H37Rv during passages.
Conclusions: GR MDR/xDR isolates of Beijing
family varied, and was higher in the untreated vs
treated patients. Passages in 8 of 10 cases did not
affect GR. Mtb GR should be considered with clinical
and strain genetic data.
P109
DORMANCY: A SYSTEMATIC STuDY
Barbara Tizzano1, Stefan Niemann1
1
borstel, Germany
Mycobacterium tuberculosis, one of the major
responsible of mortality worldwide, has the ability to
latently reside in humans for decades without causing
clinical symptoms. The mechanisms leading to this
condition are not entirely elucidated. The Wayne
hypoxic model is one of the most successful models
so far described to investigate the “dormant” state, a
bacterial phenotype characterized by slow replication,
increased resistance to antibiotics and loss of
metabolic activity. Wayne and coworkers showed that
Mycobacterium tuberculosis can survive anaerobiosis
for long periods of time only after a gradual transition
to a microaerophilic stationary phase. The Wayne
model has been so far employed to investigate
dormancy in H37Rv but the knowledge on hypoxiainduced dormancy in other phylogenetic branches of
Mycobacterium tuberculosis is still scarce.
Based on the hypothesis that different lineages may
play different roles in disease outcome, vaccine
efficacy, drug resistance and biological responses
in experimental models, our study focuses on the
dormancy phenotype induced by anaerobic conditions
and we analyzed lineage-specific differences in the
transition from the active state to non-replicating
persistence. With this aim we set up the Wayne model
for clinical isolates belonging to Euro American, East
African Indian and Beijing lineages. Dormancy was
induced in the laboratory strain H37Rv, two clinical
isolates of the Haarlem lineage, two from Cameroun,
three from East African Indian and three from Beijing.
Further goal of our work was to investigate reactivation
responses in the selected strains and to identify
transcriptional changes occurring in early stages of
resuscitation. In this study reactivation triggered by
oxygen and fresh medium is showed.
In conclusion, we have set up a dormancy model
with strains from different lineages of Mycobacterium
tuberculosis to investigate the lineage-specific
responses to hypoxia and the ability to survive and
recover from this stress condition. Typical growth
curves were obtained by OD594 and confirmed by
c.f.u. Our results indicate that all the strains belonging
to the Euro American phylogeny are able to survive
the anaerobic conditions and they recover their
ability to grow and divide in liquid and solid medium.
Interestingly, the East African Indian and the Beijing
lineages do not survive anaerobiosis and they lose
the ability to resuscitate. Microarray analysis on RNA
sampled from different strains and at different time
points will reveal more about the mechanisms behind
dormancy and reactivation.
P210
RECOGNITION OF ZnT8, PROINSuLIN
AND hOMOLOGOuS MAP PEPTIDES IN
SARDINIAN ChILDREN AT RISk OF T1D
PRECEDES DETECTION OF CLASSICAL ISLET
AuTOANTIBODIES
Leonardo A Sechi1, Magda Niegowska1, Daniela
Paccagnini1
1
university of sassari
Antibodies recognizing mycobacterium avium
subspecies paratuberculosis epitopes cross-react
with the β-cell antigens zinc transporter 8 (zNt8) and
proinsulin in type 1 diabetes patients.
As numerous evidences highlight the increasing
incidence of type 1 diabetes mellitus (t1D) in children,
an early diagnosis is crucial for defining correct
treatment and diet. Recent studies suggest that
detection of antibodies against znt8 in pre-clinical
phase can predict t1D development. Previously,
we demonstrated a significative association of
Mycobacterium avium subspecies paratuberculosis
(MAP) with t1D in adult Sardinian patients. to
enforce this finding, we investigated the presence
of antibodies against znt8 and proinsulin, with
respective MAP homologous peptides associated to
t1D, in children at risk for t1D involved in the tRIGR
study. Positivity was observed in 48% of patients
compared to 5,85% of healthy controls, preceding
appearance of anti-islet autoantibodies. Being MAP
easily transmitted to humans with infected cow’s milk
and detected in retail infant formulas in Europe, MAPderived peptides could be present in untreated and
hydrolysed formula, and act as antigens stimulating
β-cell autoimmunity.
77
TB EPIDEMIOLOGY
P30
GENOTYPING OF MuLTIDRuG RESISTANT
MYCOBACTERIuM TuBERCuLOSIS ISOLATES
IN jALISCO MExICO
Gladys Lopez-Avalos1, Martin Lopez-Rodriguez2,
Manuel Sandoval-Diaz 3, juan Carlos VillanuevaArias 3, Carlos Vázquez-Chacon4, Ikuri AlvarezMaya1
1
the center for research and Applied
technology in Jalisco (ciatej)
2
Public Health laboratory of Jalisco
3
Health Minister of Jalisco
4
institute for epidemiologic Diagnosis and
reference (indre)
Tuberculosis is a disease caused by the bacillus
Mycobacterium tuberculosis, the transmission of
M. tuberculosis presumably fuelled the worldwide
problem of emerging drug resistance. One of the
biggest concerns in TB control is the dispersion of
strains resistance to first line antibiotics. there is
evidence that the genetic variability among clinical
isolates may have consequences on the outcome of
infections. In México, especially in the state of jalisco,
there are few studies on the dispersion of tuberculosis
strains. To analyze the pattern of tuberculosis
strains in Jalisco we studied the genetic diversity to
understand the dynamics of transmission in Mexico.
This study was conducted with isolates from the Public
Health Laboratory of jalisco, collected from july
2012 to July 2013. A combination of both methods
Spoligotyping (Diagnostic kit ocimum Biosolutions)
and 15 MIRu-vNtR were assesses. out of a total of
65 isolates tested by drug susceptibility 26.5% were
sensitive to the first line drugs, 7.8% were classified
as multi drug resistant, 26.5% were mono-resistant,
14.0% poly-resistant and 25% were patients who
received treatment for 6 months but the treatment
failed. The spoligotyping detected different types of
strains linages in jalisco, t1 (18.7%), H3 (7.8%),
MANu (4.6%), x1 (3.1%), EAI5 and LAM1 (1.5%),
finally orphan strains were the bigger group found
in jalisco (60%). Dendogram analysis indicated that
none of the multidrug resistant isolates had a similar
MIRu-vNtR pattern. the analysis will be useful to
the health sector to control resistance to first line
antibiotics. this study was supported by CoNACyt
grant SALuD-2012-01-180574.
78
P51
MONITORING OF MYCOBACTERIuM BOVIS
FROM FREE-RANGING WILDLIFE IN SOuTh
kOREA
Yun-ho jang1, Soyoon Ryoo1, Yoonra jang1,
Narae kim1, jin kyoung kim1, hang Lee2, Soyoung Park3, Woong-seog Song3, jong-Taek
kim4, Soo hee Lee1, jae Myung kim4*
1
Animal, Plant and Fisheries Quarantine and
inspection Agency
2
conservation Genome resource bank for
Korean Wildlife and research institute for
Veterinary science, seoul National university
3
Gyeonggido Northern livestock & Veterinary
Medicine, North-branch
4
section of Wildlife internal Medicine, Kangwon
National university
*corresponding Author
Bovine tuberculosis (btB), caused by Mycobacterium
bovis is a transmissible disease, that mainly affects
deer and livestock such as cattle. Bovine tuberculosis
has been identified in a wide variety of free-ranging
wildlife. Recently, awareness has increased of
tuberculosis in wildlife as a potential reservoir of
infection for domestic animals worldwide. Between
2012 and 2014, we attempted to isolate M. bovis
from 620 wild animals, mainly korean water deer
and raccoon dogs from 10 regions in South Korea.
Mycobacterium bovis was isolated in two wild boar
captured in Gyeonggi Province. Above this, M.
intracellulare was isolated from 8.06% and M. avium
subspecies avium was isolated from 1.94% of the
animals. Other non-tuberculosis materials were
isolated from 19.3% of the animals. Spoligotypes of
two M. bovis isolates were SB0140, SB1040, and
MIRu-vNtR types using 12 primers (MIRu4, MIRu16,
MIRu27, MIRu31, EtR-A, EtR-B, EtR-C, quB11b,
quB26, quB3336, vNtR2401, vNtR3171) were
4-2-3-3-7-5-5-4-4-3-4-3,
5-2-3-3-7-5-5-4-3-10-5-2.
Compared to results from near livestock farms with
bovine tuberculosis, one wild boar shared a same
pattern with this. our study shows the first case of
M. bovis infection in wild boars in South korea, and
transmission between domestic animals and wild boar
would be possible. Although number of wild boars used
this study is not enough to overall epidemiological
situation of btB in South korea, because of their high
densities and activity ranges, wild boars have the
potential to spread M. bovis across a broad range of
animals, including wild and domestic animals. Further
studies are needed to improve the understanding of
tuberculosis epidemiology in wild animals and assess
the interspecies transmission between wild and
domestic animals in South Korea.
P67
P84
MOLECuLAR SNAPShOT OF MYCOBACTERIuM
TuBERCuLOSIS POPuLATION IN REPuBLIC OF
kARELIA, RuSSIAN FEDERATION
Anna Vyazovaya1, Natalia Solovieva2,
julia kononenko3, Natalia Melnikova2,
Tatiana Sunchalina3, Daria Starkova1, Igor
MOkROuSOV1, Viatcheslav Zhuravlev2, Olga
Narvskaya4
1
2
3
republican tb Dispensary, republic of Karelia
4
st. Petersburg Pasteur institute, research
institute of Phthisiopulmonology
PRACTICAL APPLICATION OF MOLECuLAR
GENOTYPING OF MYCOBACTERIuM
TuBERCuLOSIS FOR ThE TuBERCuLOSIS
CONTROL IN ThE COuNTRY
Iveta Ozere1, Inta jansone2, Ilva Pole2, Girts
Skenders3, Anda Nodieva4, Anita Skangale3,
Olga Bobrikova3, Zita Lauska3, Ruta Pastare5,
Nina Gusarevica6, Margarita Baumane7,
Matiss Bauskenieks2, Renate Ranka2, Viesturs
Baumanis2
1
Riga Stradiņš University; Clinic of Tuberculosis
and lung Diseases, riga east university
Hospital, latvia
2
latvian biomedical resarch and study centre,
riga; clinic of tuberculosis and lung Diseases,
riga east university Hospital, latvia
3
4
Riga Stradiņš University, Latvia
5
balvu and Gulbene Hospital Association, latvia
6
Daugavpils regional Hospital, latvia
7
tukuma Hospital, latvia
Background: Russian Republic of Karelia is located
on Russian-Finnish border and includes most of
historical karelia land. In 2014, tuberculosis (tB)
incidence here decreased since 2009 (45.8 vs 62.3 /
100 000). We performed a molecular characterization
of Mycobacterium tuberculosis isolates obtained in
Republic of Karelia in 2013-2014.
Methods: M. tuberculosis isolates were recovered
from newly-diagnosed pulmonary TB patients. Drug
susceptibility testing was done with method of absolute
concentrations. DNA was subjected to spoligotyping,
spoligotypes were compared to SItvIt_WEB and
MIRu-vNtRplus. LAM family was detected by testing
specific Rv0129c SNP, LAM isolates were tested for
RD115, RD174, RD-Rio, and LAM-RuS markers.
Beijing B0/W148 cluster was identified by testing
Rv2664-Rv2665::IS6110 insertion.
Results: Spoligotyping of 74 M. tuberculosis isolates
revealed 24 profiles including 7 shared types (2 to 40
isolates) and 15 singletons. the largest types were
SIt1 (Beijing, n=40) and SIt40 (t4, n=5). Beijing
family was the largest (n=41) followed by ill-defined t
family (n=11), ural (n=8) and LAM (n=8). Beijing В0/
W148-cluster was identified in 14 (34%) of 41 Beijing
isolates; all В0/W148 isolates were drug-resistant.
Seven of 8 LAM isolates belonged to RD115/LAMRuS branch, one SIt20 isolate was of RD-Rio
sublineage. MDR was found in 31 (42%) isolates and
was combined to resistance to: StR (n=31), EMB
(24), kAN (16), AMk (9) and CAP (7). MDR was
more prevalent among Beijing (25 of 41, 61%) vs
non-Beijing isolates (6 of 33, 18%; P<0.0001). three
isolates (all – Beijing) were xDR. All SIt40 isolates
were drug sensitive.
Conclusions: Circulation of the MDR associated
isolates of the Beijing genotype (55% of studied
collection) and its B0/W148 cluster (34% of the Beijing
group) continues to influence the current situation with
TB control in northwestern Russia including Republic
of Karelia. The other well-delineated genetic families
such as ural and LAM appear to be dominated by
pansusceptible isolates.
Acknowledgements: Russian Science Foundation
(project 14-14-00292).
Introduction: Latvia still belongs to high-incidence
countries with tuberculosis (tB) notification rates
of 31.6 per 100,000 in 2014. tB can be caused
by genetically various strains of Mycobacterium
tuberculosis (Mt) associated with different virulence
and drug susceptibility. Investigation of Mt strains
causing local outbreaks can be used as a tool for TB
control in a country. Spoligotyping could be applied
as a rapid and easy method for initial evaluation of
mycobacterial isolates for TB outbreak assessment.
Goal of the study: Identification of the genotypes of
Mt strains causing tB outbreaks and evaluation of
suitability of spoligotyping method for the investigation
of acute outbreaks.
Materials and methods: Retrospective crosssectional study. Spoligotyping was applied to Mt
isolates obtained from children and their potential
sources of infection, as well as from other tB patients
that epidemiologically linked either to diseased
child or possible source case. Identical clusters of
Mt spoligotypes were assumed as an evidence
of transmission of the infection within the group of
epidemiologically linked individuals.
Results: Mt spoligotyping was performed during
2001- 2014 on Mt isolates obtained from 105 children
and their 131 potential infectious source cases.
Identical genotypes from a child and one or more
infectious sources were detected in 73/105 (70%)
of matched child-infectious source case groups. Out
of them, 13 clusters with 3-9 isolates of identical
spoligotypes in epidemiologically linked groups of
patients were identified. Spoligotypes SIt766, SIt50,
SIt42, SP57, and SIt1 were identified in biggest
clusters that included 6-9 TB isolates with identical
79
genotypes. SIt766 was identified in a large group of
9 epidemiologically linked patients. SIT50 spoligotype
was detected in a group of 7 patients, thus allowed
us to trace the transmission of the infection from the
household to the community during the three year
period. SP57 spoligotype was identified in a group
of 6 patients and transmission of the infection was
followed up during 2002-2013. Other spoligotypes
causing outbreaks were SIt53, SIt254, SIt262,
SIt40 and unique spoligotypes. In outbreaks related
to SIt766, SIt50, SIt42, SP57, SIt1, and SIt262 the
infection was transmitted to the household contacts
and close contacts (accordingly to WHo definitions)
as well.
Conclusions:
1. Spoligotypes of Mt causing largest tB outbreaks
might be assumed as potentially more virulent in
terms of the disease in contacts
2. Prophylactic interventions and contact investigations
must be more aggressive for Mt with spoligotypes
SIt766, SIt254, SIt1,SIt42 and SP57
3. Routine use of spoligotyping of Mt in clinical
practise is suitable method helping clinicians to get
initial orientation in TB outbreak
Acknowledgments: This study was supported by
the vPP 2014 “BIoMEDICINE”
P100
LABORATORY CROSS CONTAMINATION
– MOLECuLAR EVIDENCE OF FALSEPOSITIVE CuLTuRES OF MYCOBACTERIuM
TuBERCuLOSIS
Ewa Augustynowicz Kopeć1, Monika Kozińska1,
katarzyna Wasiak1, Anna Borek1, Dagmara
Borkowska1, Magdalena klatt1
1
research institute of tuberculosis and lung
Diseases
Identification of Mycobacterium tuberculosis complex
in clinical material from the patient is the basis of
microbiological confirmation of tB, and remains gold
standard for diagnosis. The results of numerous
studies show that about 0.1-4% derived culture
may be the result of cross-contamination. It occurs
most frequently during processes of collection and
processing of clinical samples, as a result of infection
during medical procedures. The misdiagnosis of M.
tuberculosis infection has many consequences medical and psychological implications for patients
and their families and financial and public health
implications for health-care institutions. Microbiology
laboratory procedures should minimize the possibility
of laboratory cross-contamination of specimens and
maximize the ability to recognize a cluster of falsepositive results. Molecular typing methods provide
rapid, accurate and effective means of identifying
false-positive M. tuberculosis cultures.
Aim of study: Determining the incidence of cross-
80
contamination in the National Tuberculosis Reference
Laboratory in Poland during 2011 - June 2014.
Materials and Methods: The material for the study
included 598 isolates of M. tuberculosis complex from
408 patients with suspected tuberculosis. For the
epidemiological and molecular analysis we selected
26 (4.3%) cultures, which, according to the CDC
guidelines, could be potentially false-positive. the
information that determined the choice of strains is the
same date of inoculation and the sequence number
of the study or in close proximity to the material from
which the culture was obtained. Molecular analysis
of the strains was performed using spoligotyping and
MIRu-vNtR methods.
Results: We examined 12 potential crosscontamination incidents. On the basis of DNA
fingerpinting, contamination was confirmed in 4
cases. the consequence was to obtain 5 (0.8%)
false-positive cultures of M. tuberculosis complex.
Conclusions: Detection of cross-contamination of
materials from patients with suspected tuberculosis
has epidemiological, clinical and therapeutic
significance. Proper system of monitoring and
identification of false positive cultures requires
reliable medical documentation and using appropriate
genotyping methods. Good communication between
clinicians and the laboratory staff is required for the
rapid identification of specimen cross-contamination.
P101
ExPLORING ThE SOCIODEMOGRAPhIC AND
CLINICAL FEATuRES OF ExTRAPuLMONARY
TuBERCuLOSIS IN SAuDI ARABIA
Sahal Al-hajoj Al-Nakhli1, Bright Varghese1
1
King Faisal specialist Hospital and research
centre, riyadh, saudi Arabia
Background: Saudi Arabia annually reports a
relatively higher proportion (28-32%) of extrapulmonary
tuberculosis (EPtB) cases in comparison to other
global regions. However, there were few studies
conducted so far to determine the sociodemographic
factors and clinical manifestations associated with
EPTB at a nationwide level.
Methodology: A retrospective analysis on culture
positive EPTB isolates collected from all the
provinces of the country were conducted for a period
of 12 months to determine the spectrum of diversity
in EPTB infection sites and the confounding factors.
A detailed clinical and demographical data analysis
was carried out along with first line drug susceptibility
testing.
Principal findings: Intra-thoracic and extra-thoracic
lymph nodes (44.6%) were the most common sites
of infection followed by gastrointestinal (17.3%) and
central nervous systems (11.8%). Male patients were
mostly infected (58.8%), in contrary to the global trend.
Any drug resistance was observed in 23.1% isolates
with a 2.1% of multi-drug resistance. HIv reactivity
was found only in 2.2% cases. A higher proportion of
Saudi nationals (58.8%) were infected compared to
the immigrants, descending mostly from South Asia
(34.4%) and South East Asia (31.2%). the Saudi
population predominated with all forms of EPTB while
immigrants showed no significant variations.
Conclusions: Saudi Arabia faces a serious threat
from EPtB, particularly to the central nervous
system and gastrointestinal systems. More effective
diagnostic strategies and control measures must be
implemented to reduce the high rate of EPTB in the
country. In addition, these findings warrant further
detailed research to explore all related comorbid
conditions of EPtB development, particularly the
host-related factors.
P142
DIRECT SPOLIGOTYPING ON DNA ExTRACTED
FROM MYCOBACTERIuM TuBERCuLOSIS
POSITIVE ZIEhL-NEELSEN STAINED SLIDES
FROM EThIOPIA
Barbara Molina-Moya1, Silvia Blanco2, Mulualem
Agonafir3, Michel Gomgnimbou4, Lizania
Spinasse5, Meissiner Gomes1, Guislaine
Refrégier5, Daniel Datiko6, Luis Cuevas7, jose
Dominguez1, Christophe Sola5
1
2
Department of Microbial, cellular, and Molecular
biology; college of Natural sciences, Addis
Ababa university
3
Nfectious and other Diseases research
Department; ethiopian Health and Nutrition
research institute, Addis Ababa
4
orsay, France; centre Muraz, bobo-Dioulasso,
burkina Faso
5
orsay
6
lstm, university of Hawassa
7
tuberculosis (tB) remains one of the most threatening
diseases. Ethiopia has the second largest population
in Africa and ranks 7th among the high TB burden
countries. The aim of this work was to perform
high-throughput spoligotyping of Mycobacterium
tuberculosis strains isolated from TB patients in
Ethiopia. A total of 93 sputum specimens were
collected and examined by ziehl-Neelsen staining
and smear microscopy. DNA was extracted from the
stained preparations and analyzed by spoligotyping
with a microbead-based method in a Luminex
device. Interpretation of raw hybridization data was
done by two experts. The 93 sputum specimens
analyzed were smear 2+ or smear 3+. Interpretable
spoligotyping results were obtained in 60 of the 93
DNA extracts analyzed, yielding a genotyping rate of
64.5%. the following global spoligotyping distribution
was observed: 25 patterns belonged to the t1 family,
11 patterns to t3-EtH, 11 patterns to H3, and the
remaining 13 patterns to other clades. The 25 patterns
classified as t1 were distributed in five clusters (SIt
53, n=10; SIt 245, n=7; SIt 1877, n=3; SIt 2447,
n=2; SIt 373, n=2) and one pattern was found to be
orphan. The 11 patterns belonging to T3-ETH were
clustered under the SIT 149. Among the 11 patterns
classified as H3, a single cluster was found (SIt 777,
n=5) and six patterns were orphan. Finally, the genetic
diversity of the remaining 13 patterns was observed
as follows: CAS1-kili (SIt 21, n=2); MANu2 (SIt
1096, n=2); SIt 46, n=2; and seven orphan patterns.
In addition to the spoligotyping results, we attempted
to genotype SNPs in genes related to drug resistance
(rpob, katG, inha). However, no interpretable results
were obtained due to the poor quality of DNA. In
conclusion, to our knowledge, this is the first time
direct spoligotyping is performed on DNA extracted
from ziehl-Neelsen-stained slides in Ethiopia, by
a simple and high-throughput method, and with an
acceptable genotyping rate, providing an alternative
way to accurently rapidly identify TB patients by their
spoligotyping profile.
P149
GENETIC DIVERSITY OF MYCOBACTERIuM
TuBERCuLOSIS IN NIGERIA BASED ON
SPOLIGOTYPING ON DNA ExTRACTED FROM
POSITIVE ZIEhL-NEELSEN-STAINED SLIDES
Barbara Molina-Moya1, Silvia Blanco2, Michel
Gomgnimbou3, Lizania Spinasse4, Meissiner
Gomes1, Guislaine Refrégier4, Luis Cuevas5,
Lovett Lawson6, Saddiq T. Abdurrahman7, joshua
Obasanya7, jose Dominguez1, Christophe Sola4
1
2
Department of Microbial, cellular, and Molecular
biology; college of Natural sciences, Addis
Ababa university
3
orsay; centre Muraz, bobo-Dioulasso, burkina
Faso
4
orsay
5
6
bingham university, Nassarawa state
7
National tb, buruli ulcer and leprosy control
Programme, Abuja
tuberculosis (tB) remains one of the most threatening
diseases. Nigeria, the most populous country in
Africa, has one of the world’s largest burdens of
tB. our objective was to perform high-throughput
81
spoligotyping and detection of mutations conferring
drug resistance in Mycobacterium tuberculosis strains
circulating among patients with suspected drug
resistance in Nigeria. A total of 929 sputum specimens
were collected in state hospitals from June 2013 to
December 2014. ziehl-Neelsen staining and smear
microscopy examination were performed. DNA was
extracted from the stained preparations. Simultaneous
spoligotyping and detection of mutations associated
with drug resistance were performed by a microbeadbased method in a Luminex device. Of the 929 sputum
specimens analyzed, 505 were smear 3+ and 424 were
smear 2+. Interpretable spoligotyping results were
obtained for 549 of the 929 DNA samples analyzed
(59.1%). Conversely, no results were obtained for
the detection of drug resistance mutations. Among
the interpretable spoligotypes, the Cameroun family
(SIt61 and derived, signature: spacers Δ23-25 and
Δ33-36) represented broadly one out of two patterns,
suggesting that it is an emerging genotype family in
Nigeria. M. africanum, which includes M. africanum
West-African 1 (SIt431 and SIt338, signature:
spacer Δ8-12 and Δ37-39) and M. africanum WestAfrican 2 (SIt181, signature: spacers Δ8-9 and Δ39),
represented one out of five patterns. this relatively
high percentage of M. africanum, compared to some
recent results obtained in Cameroun, confirms that
these strains remain broadly spread, and likely
transmitted into the Nigerian population. Finally, all
other lineages, including Euro-American (t, Haarlem,
LAM, S, etc, signature: spacer Δ33-36), and all other
M. tuberculosis signatures represented broadly
one out of three patterns, indicating that there is an
important part of the TB endemia in Nigeria due to
the Euro-American (i.e. «modern») strains. Due to
the quality of DNA, which was extracted from stained
microscopy slides, it was not possible to obtain
molecular drug resistance results. In summary, we
present an update of the M. tuberculosis spoligotypes
circulating in Nigeria. We also show that spoligotyping
data can be obtained in a rapid and high-throughput
manner with DNA extracted from zN-stained slides.
This may potentially help improving the management
of TB patients.
P163
CLONAL COMPLExITY IN MYCOBACTERIuM
TuBERCuLOSIS IS MORE ThAN AN
ANECDOTAL FINDING: 2 ExAMPLES OF hOW
IT ChALLENGES STANDARD DIAGNOSTIC
PRACTICE
Laura Pérez-Lago1, Miguel Martínez Lirola2,
Yurena Navarro1, Marta herranz1, María jesús
Ruiz Serrano1, Pilar Miralles1, Emilio Bouza1,
Darío García de Viedma3
1
Hospital General universitario Gregorio
Marañón
2
complejo Hospitalario torrecárdenas
3
servicio de Microbiología clínica y
82
enfermedades infecciosas. Hospital General
universitario Gregorio Marañón; inst.
investigación sanitaria Gregorio Marañón; cei
campus Moncloa, ucm-upm, Madrid, spain
Mixed infections by MtB are expected in high
prevalence areas but not considered outside these
areas. We present 2 examples of complex infections
that challenged standard diagnostic procedures. The
first involved a patient coinfected by a susceptible
strain and an under-represented MDR strain. the
genotypic susceptibility test results were ambiguous.
MIRu-vNtR revealed double alleles in 11 loci,
thus confirming coinfection by 2 strains. Analysis of
independent colonies made it possible to separate the
strains and showed that the MDR strain was a minority
strain (2%). the MIRu pattern for the MDR strain was
identical to that of an MDR case the patient had been
in close contact with 3 years previously. The pattern
for the susceptible coinfecting strain was also found
in another 4 cases in the same area (years 2009,
2011 [2] and 2014), indicating that it was a circulating
strain. This event was most likely a reactivation of a
latent MDR strain that coincided with a recent infection
by a susceptible strain. The second example was an
alert of a false tB diagnosis involving 2 HIv-positive
cases hospitalized at the same unit. One case (case
A) accumulated 12 MtB-positive specimens whereas
only 1 of 3 specimens for the other case (case B,
potentially false positive) was MtB-positive. A fast
MIRu-vNtR analysis (standard electrophoresis)
revealed 2 different patterns, suggesting true tB in
both cases. However, lack of clinical support and
a final negative result for the 2 cultures in case B
necessitated an in-depth analysis. MIRu-vNtR
performed directly from 10 sputa from case A and
subsequent capillary electrophoresis revealed the
coexistence of 2 coinfecting strains: one an overrepresented strain (strain A) and another clearly a
minority strain that corresponded to the strain (strain
B) in the positive specimen wrongly assigned to case
B. Differences in the representativity of the strains in
the sequential sputa from case A were tracked, and
some specimens were enriched only with one strain.
The most likely explanation for this event was that the
false positivity alert was resolved erroneously owing
to involvement of a true-positive case with mixed
infection by 2 MtB strains whose representativity
differed in several samples. Clonal complexity in
MtB must be borne in mind in clinical practice, even
in areas of moderate incidence, to correctly interpret
challenging situations. Funding: Plan Estatal I+D+I
2013/16, ISCIII (PI12/02080, 13/01207) and FEDER
P166
INTERLABORATORY EVALuATION AND
OPTIMIZATION OF qIAxcel ADVANCED SYSTEM
FOR MIRu-VNTR TYPING OF MYCOBACTERIuM
TuBERCuLOSIS
Alberto Trovato1, Broda Agnieszka2, Emanuele
Borroni1, Vladyslav NIkolayevskyy2, Francis
Drobniewski2, Daniela Maria Cirillo3
1
2
infection and immunity Department, Medical
school, imperial college london; blizard
institute of cell and Molecular science, Queen
Mary university of london, united Kingdom
3
Diseases, San Raffaele Scientific Institute, Milan,
italy
The 24-locus mycobacterial interspersed repetitiveunit-variable-number tandem repeat (MIRu-vNtR)
genotyping is an effective and reliable standardized
method in Mycobacterium tuberculosis molecular
epidemiology. However, manual methodology, the
most used in resource-limited settings, is by far
more prone to errors and misinterpretation than the
automated technique, utilizing capillary fragment
separation on sequencers.
Recently, the qIAxcel Advanced System (qiagen
AG, Germany), a capillary electrophoresis-based
technology, has been proposed as a cost-effective
alternative to both manual and automated technique.
Our study focused on the assessment of the accuracy
and interlaboratory reproducibility of this technology.
A total of 60 reference strains (1440 loci) were tested
for 24-locus MIRu-vNtR both at Imperial College
(London) and San Raffaele Scientific Institute (Milan)
by using two slightly different run protocols, namely
oM1700 and oH1700 respectively, and a common
size marker (100–2500 bp).the results were then
compared to reference values.
The overall concordance to reference values was
98.89% for samples tested in London and 98.54%
for those analysed in Milan. very similar results were
reported by the two laboratories with some important
differences for locus 1644 (MIRu 16) and 2165
(EtRA).
Interestingly, both laboratories reported 4052 as the
most problematic locus, probably as the system tends
to overestimate the size of high molecular weight
amplicons. Apparently, locus 960 was the only one to
show very precise size estimation even over 1000 bp.
Both London and Milan reported 100% concordance
for fragments under 700 bp.
Two different approaches were followed to improve
the system performance: (i) the development of an inhouse marker, and (ii) modifications of calling tables
with offset values to reflect variations in the PCR
fragment mobility under denaturation conditions.
The in-house marker was developed by using as
reference marker a sample with pronounced stutter
peaks at locus 4052 or by mixing together different
amplicons. By using stutter peaks a good improvement
was obtained but limited to locus 4052 itself. By
putting together different fragments we didn’t obtain
satisfactory results. Conversely, the introduction of
modified calling tables with offset values, using the
commercial size marker, showed the best results.
In conclusion, the qIAxcel Advanced System is a
valid alternative as automated system for genotyping
M. tuberculosis.
P169
DISTINCT MYCOBACTERIuM TuBERCuLOSIS
SPOLIGOTYPES-RELICTS OR INVASIVE
STRAINS IN LATVIA
Ilva Pole1, Girts Skenders2, Inta jansone3,
Viktorija Igumnova3, janis Pjalkovskis3, Anda
Nodieva4, Iveta Ozere5, Vija Riekstina2, Renate
Ranka3
1
centre of tuberculosis and lung Diseases, riga
east university Hospital; latvian biomedical
research and study centre
2
3
latvian biomedical research and study centre,
riga, latvia
4
Riga Stradiņš University, Riga, Latvia
5
clinic of tuberculosis and lung Diseases,
riga east university Hospital; Department of
Infectology and Dermatology, Riga Stradiņš
university, riga, latvia
Introduction: tuberculosis (tB) is a widespread
and ancient disease in Eastern Europe. Although the
epidemiological situation of TB is slightly improving
every year, it is still one of the major infectious
diseases that cause serious health problems. Ordinary
Mt genotyping by spoligotyping method revealed
that most common Mycobacterium tuberculosis
(Mt) isolates observed in Latvia belonged to the two
main Mt lineages: East-Asian (~26%) and EuroAmerican (~60%); both are well-known in Europe
and in the immediate neighborhood published in the
SItvIt database. Nevertheless, among all detected
genotypes a variety Mt isolates of rare or so far
unspecified spoligotype patterns were observed.
thus the question arises whether these Mt isolates
are relicts of the last century, or they are so far
unrecognized invasive entrants.
The aim: of this study was to characterize and
describe rare and non-published genotypes of Mt
isolates obtained in Latvia.
Materials and Methods: In this study, 93 Mt isolates
with non-published in SItvIt database spoligopattern
(SP) obtained from 2009 to 2012 in Riga and Riga
district were selected. Epidemiological relationships
83
of these Mt isolates with each other as well as with
all well specified Mt genotypes from Latvia were
studied, and obtained data were analyzed in relation
to the possible evolution of direct repeat (DR) region
and similarity of RFLP patterns.
Results: From 93 available and genotyped Mt isolates
with non-published in SItvIt database spoligopatterns
85 were sensitive, but 8 were resistant to one to
three anti-tB drugs. Within all spoligotypes studied,
43 isolates (46%) formed 11 different clusters with 2
– 11 members. Remaining 50 samples (54%) were
individual with the unique spoligo and RFLP pattern.
Searches of clusters of Mt genotype across the Latvia
showed some small infections foci of tB outside Riga;
however the true history remains
unclear.
Conclusions:
1. the question remains open whether the Mt strains
with non-published genotypes were endemic or of
the outside origin.
2. Most of observed tB genotypes were diverse,
representing just a few disease`s cases.
3. Existence of spoligotype clusters requires strong
epidemiological tracing.
disease. M. tuberculosis isolates from 22 pairs of
patients living in the same household, and first and
last isolates from 8 patients with an extended period
of disease were selected. Based on the hypothesis
that SNP identification provides a measure of genome
evolution over time, we calculated pairwise genomic
differences between the respective pairs of isolates.
The SNP variation detected between household
contacts were greater than anticipated despite
closely related IS6110 DNA fingerprints. this study
highlights genomic instability during transmission and
persistent disease thereby questioning whether the
method used to infer transmission chains should be
revised for high tuberculosis incidence settings. In
this study, we highlight the difficulties of establishing
thresholds to infer direct epidemiological links. Our
results provide a foundation for the application of
whole genome sequencing data to the elucidation of
microevolution during transmission.
Acknowledgments: This study was supported by
the ESF/1.1.1.2.0/038 and vPP “BIoMEDICINE”.
WhOLE GENOME SEquENCE OF
MYCOBACTERIuM SuRICATTAE, ThE MAIN
CAuSE OF TuBERCuLOSIS IN MEERkATS
(SuRICATA SuRICATTA)
Anzaan Dippenaar1, Sven Parsons2, julian
Drewe3, Abdallah Abdallah4, Ruben van der
Merwe2, keith Siame2, Alan Christoffels5,
Samantha Sampson2, Nico Gey van Pittius2, Paul
van helden2, Rob Warren2, Arnab Pain4
1
Faculty of Medicine and Health sciences,
Division of Molecular biology and cellular
biology, stellenbosch university
2
stellenbosch university
3
royal Veterinary college, london
4
King Abdullah university of technology
5
south African National bioinformatics institute
P176
INVESTIGATING MICROEVOLuTION OF M.
TuBERCuLOSIS DuRING TRANSMISSION
Anzaan Dippenaar1, Margaretha de Vos2, Ruben
van der Merwe2, Gian van der Spuy2, Rob Warren
2
, Samantha Sampson2, Paul van helden2, Arnab
Pain3
1
Faculty of Medicine and Health sciences,
Division of Molecular biology and cellular
biology, stellenbosch university
2
stellenbosch university
3
King Abdullah university of technology
Recent studies have shown that Mycobacterium
tuberculosis populations infecting the host are more
heterogeneous than previously considered. The
phenomenon of a subpopulation of clonal variants
and the impact of a transmission event on the
genomic stability of the infectious M. tuberculosis
population remain largely unexplored. Due to
decreasing costs and unprecedented resolution,
whole genome sequencing is an ideal tool to study
the microevolution of M. tuberculosis; it could
potentially be applied to study outbreaks in high
tuberculosis burden settings where epidemiological
data are hard to obtain. The study of microevolution
during disease and transmission is crucial to better
understand outbreak dynamics in a high tuberculosis
incidence setting where multiple sources of infection
exist. Here, we use whole genome sequencing to
investigate microevolution in M. tuberculosis during
household transmission as well as during persistent
84
P179
Tuberculosis occurs in various mammalian hosts and
is caused by a wide range of Mycobacterium species.
A recently described species, M. suricattae, has been
reported to cause tuberculosis in meerkats (Suricata
suricatta) in Southern Africa. the preliminary genetic
analysis showed it to be closely related to the M.
tuberculosis complex dassie bacillus. Here, we make
use of whole genome sequencing to describe the
genome of M. suricattae, confirming known and novel
regions of difference, SNPs and IS6110 insertion
sites. To better determine its evolutionary position
in the phylogeny of the Mycobacterium tuberculosis
complex, we used genome wide phylogenetic analysis
to show that M. suricattae clusters with the chimpanzee
bacillus, isolated in West Africa. We also propose an
evolutionary scenario for the M. africanum lineage 6
complex, showing the evolutionary relationship of M.
africanum and chimpanzee bacillus, and the closely
related members M. suricattae, dassie bacillus and
M. mungi.
P181
P182
hIGh-ThROuGhPuT MYCOBACTERIAL
INTERSPERSED REPETITIVE uNIT–VARIABLE
NuMBER TANDEM-REPEAT GENOTYPING
FOR MYCOBACTERIuM TuBERCuLOSIS
EPIDEMIOLOGICAL STuDIES
Marie Gauthier1, Floriane Bidault1, Amandine
Mosnier1, Nino Bablishvili2, Nestani Tukvadze2,
Silaphet Somphavong3, Phimpha Paboriboune3,
Oksana Ocheretina4, jean William Pape4, Glaucia
Paranhos-Baccala1, jean-Luc Berland1
1
laboratoire des Pathogènes emergents (lpe),
Fondation Mérieux-centre international de
recherche en infectiologie (ciri)
2
National center for tuberculosis and lung
Disease (Nctld)
3
centre D’infectiologie christophe Mérieux Du
laos (cicml)
4
les centres Gheskio
DETECTING PSEuDO-BEIjING M.
TuBERCuLOSIS ISOLATES uSING TBMINER
Memona Yasmin1, jérôme Azé2, Rubina
Tabassum3, Sabira Tahseen4, Shahid Abbasi5,
Naeem Akthar5, Riwan Iqbal6, Christophe Sola7,
Guislaine Refrégier7
1
Nibge, Pieas
2
lirmm
3
Health biotechnology Division, National institute
for biotechnology and Genetic engineering
(Nibge), Faisalabad, Pakistan
4
National tb reference laboratory
5
Armed Forces institute of Pathology
6
Pakistan Medical research council
7
institute for integrative biology of the cell,
université Paris-sud
The emergence of drug-resistant forms of
tuberculosis (tB) represents a major public health
concern. understanding the transmission routes
of the disease is a key factor for its control and
for the implementation of efficient interventions.
Mycobacterial interspersed repetitive-unit–variablenumber tandem-repeat (MIRu-vNtR) marker typing
is a well-described method for lineage identification
and transmission tracking. However, the conventional
manual genotyping technique is cumbersome and
time-consuming and entails many risks for errors,
thus hindering its implementation and dissemination.
We describe here a new approach using the qIAxcel
system, an automated high-throughput capillary
electrophoresis system that also carries out allele
calling. This automated method was assessed on
1,824 amplicons from 82 tB isolates and tested with
sets of markers of 15 or 24 loci. Overall allele-calling
concordance between the methods from 140 to 1,317
bp was 98.9%. DNA concentrations and repeatability
and reproducibility performances showed no biases in
allele calling. Furthermore, turnaround time using this
automated system was reduced by 81% compared
to the conventional manual agarose gel method. In
sum, this new automated method facilitates MIRuvNtR genotyping and provides reliable results.
therefore, it is well suited for field genotyping. the
implementation of this method will help to achieve
accurate and cost-effective epidemiological studies,
especially in countries with a high prevalence of
tB, where the high number of strains complicates
the surveillance of circulating lineages and requires
efficient interventions to be carried out in an urgent
manner.
tBminer is an on-line classification tool based on
spoligotype and MIRu-vNtR data. It annotates
user strains according to 3 independent and largely
used taxonomies: spoligotype-derived (SItvItWEB),
MIRu-vNtR-derived
(MIRu-vNtRPlus),
spoligotype+MIRu-vNtR-derived
(tBlineage).
When consensus between the 3 assignations exists,
TBminer makes it explicit and proposes a new
consensual taxonomy (Azé et al., 2015, submitted).
Pseudo-Beijing sublineage was discovered by Fenner
et al. in 2011. It is characterized by a spoligotype
profile identical to bona fide Beijing but associated
to a smaller deletion in the CRISPR locus. MIRuvNtR-based analysis indicates that it belongs to
CAS lineage.
We wanted to test the ability of TBminer to detect
discordance between 24 or 15 MIRu-vNtR data and
spoligotype profiles. We used a convenient sample
from Pakistan, a country known to harbor a large
diversity of CAS isolates. DNA was extracted by a
CTAB procedure or by thermolysis. We performed
43-spacers Luminex spoligotyping and 24 MIRuvNtR genotyping.
out of 225 isolates with complete genotypes, 137
isolates carried a CAS spoligotype profile (61%) and 8
isolates had a Beijing spoligotype profile (4%). other
isolates were classified as Euro-american (15%), EAI
(5%) or could not be classified by SItvItWEB (15%).
Among the 8 potential “Beijing” isolates, 6 showed a
24 and a 15MIRu-vNtR patterns typical of Beijing
lineage as analyzed independently both by TBminer
(tool predicting MIRu-vNtRplus classification) and
by MIRu-vNtRplus itself. In contrast, 2 other isolates
were classified by both tools as CAS. these two
isolates carried 2 copies of ETR-C repetition instead
of 4 as in Beijing isolates, a feature present in Fenner
et al. first Pseudo-Beijing isolates. tBminer was
thus able to provide a reliable annotation for these
isolates.
Altogether, we confirmed that in countries with high
CAS prevalence, some isolates carrying a Beijing
spoligotype may in fact be CAS isolates. In addition,
85
we show that tBminer is an efficient tool to detect
discordance between standard classifications’
signatures. The ease of use of TBminer should
help surveillance epidemiologists and clinical
microbiologists to analyze more efficiently tB genetic
diversity in their countries.
P97
DECIPhERING TuBERCuLOSIS DYNAMICS
IN EAST GREENLAND: GENOME BASED
MOLECuLAR EPIDEMIOLOGY OF
MYCOBACTERIuM TuBERCuLOSIS IN A hIGh
INCIDENCE, LOW DIVERSITY SETTING
karen Bjorn-Mortensen1, Aase Bengaard
Andersen2, karin Ladefoged3, Bolette Soborg4,
Anders koch4, Troels Lillebaek5, Thomas kohl6,
Stefan Niemann6
1
Department of epidemiology research,
international reference laboratory of
Mycobacteriology,statens serum institut,
copenhagen; Greenlands center of Health
research, Nuuk, Greenland
2
Department of infectious Diseases, copenhagen
university Hospital and university of southern
Denmark, odense, Denmark
3
Department of internal Medicine, Queen ingrid’s
Hospital, Nuuk
4
Department of epidemiology research, statens
serum institut, copenhagen, Denmark
5
statens serum institut
6
borstel, Germany
Introduction: tuberculosis (tB) is again a major
health problem in Greenland. TB outbreaks often
occur in isolated settings with complex social networks
revealed by contact tracing. This complexity makes
it difficult to identify cases of active Mycobacterium
tuberculosis (Mtb) transmission in order to confine
the outbreaks. TB incidence in the isolated East
Greenland has increased dramatically in recent
years to approximately 1,000 notified tB cases per
100,000 inhabitants and approximately 40% of the
East Greenlandic population is infected with Mtb.
Since 2004, all culture-positive tB samples from East
Greenland have been genotyped with mycobacterial
interspersed repetitive unit-variable number of tandem
repeats (MIRu-vNtR). Available data showed that
almost all strains were from two major clusters with
very similar MIRu-vNtR genotypes, making it difficult
to distinguish new from old cases.
Objective: The study aimed to characterize
transmission patterns in a population-wide whole
genome sequencing (WGS) study including all
culture-positive cases within a 21-year period. The
main objective was to determine to what extend WGS
contributes to the understanding of an outbreak in a
high tB incidence, low diversity setting.
86
Methods: using Illumina Nextera xt library preparation
kits and the NextSeq500 next generation sequencing
platform, we performed WGS of 182 isolates (98%
of culture-positive cases). After reference mapping to
the H37Rv genome, detected variants were combined
to yield a set of phylogenetically informative single
nucleotide polymorphism (SNP) positions.
Results: A maximum parsimony tree based on
these positions revealed the same pattern as seen
with MIRu-vNtR, but with a much higher resolution.
Employing a maximum distance of 12 SNPs between
isolates, four groups were created, which overall
correlated well with the MIRu-vNtR clustering. Both
time and place of notification correlated well with WGS
data, and transmission links suggested by WGS fit
well with expansion over time. Also, epidemiological
data and WGS data together suggest a very clear
geographical distinction of clones. overall, WGS
based genotyping describes in detail the longitudinal
transmission pattern of tuberculosis in an isolated,
high incidence, low variance setting. therefore, even
among isolates with the same, or almost identical,
MIRu-vNtR type in a high incidence setting, WGS
performs well and offers a much higher resolution
than previously used genotyping methods.
2013. Based on Spoligotyping, 107 regional strains
were grouped into 18 clusters (%97,3) and 3 strains
were observed as orphan (% 2,7). t1 cluster was
included the large number of strains with 53 samples
(%48,2). Clonality analysis demonstrated that there
were no clonal relations between these strains.
Although variable repeat numbers in clusters which
determined with IS6110-RFLP could further enhance
the descriminative power, this method did not produce
significant results when used alone. As a result of
that, spoligotyping and 15 loci MIRu-vNtR were
more effective when used together for identification
of species, detection of clonal relatives and collecting
data for surveillance studies.
In conclusion, incidence rate of MDR-tB strains
which do not show clonal relations increases in
Cukurova Region of turkey. For this reason to control
and treat new MDR-tB strains, combining of these
three methods which have high discriminative power
should be use after diagnosis.
keywords: IS6110, MIRu-vNtR, Mycobacterium
tuberculosis, Spoligotyping
genotype families causing tB in Pará. MtB isolates
were obtained from clinical specimens collected
between 1998 and 2010 at the Evandro Chagas
Institute, Belém do Pará, Brazil. the DNA extraction
and spoligotyping were performed, as previously
described, according to standard protocols. Among
the 980 samples, a total of 249 distinct spoligopatterns
were observed. of these, 6.4% (63) corresponded to
orphan patterns, while 93.5% (917) belonged to 186
shared-types (SIts). Sixty-one newly created SIts
were also found (4063 to 4090). the main families
found in this study were LAM (41%; 402), t (18,9%;
185), Haarlem (12,9%; 124) and East African Indian
- EAI (9.9%; 97), followed by x (8.4%; 92), unknown
(5.7%; 56), Manu (1.8%; 18), S (0.9%; 9), Central Asian
- CAS (0.6%; 6) and ural (0.1%; 1). the predominant
lineages LAM, t and Haarlem have been previously
well described in Brazil. this study, however, shows
an emergence of EAI lineage, which comes originally
from India and Africa. It thereby provides new insight
into the population structure of MtB isolates in Pará
over a 12-year period.
P200
P204
P195
SPOLIGOTYPES OF Mycobacterium tuberculosis
ISOLATES FROM PATIENTS RESIDENTS IN
STATE OF PARá, BRAZIL
Emilyn Conceição1, Maria Luiza Lopes2, Ismari
Perini Furlaneto3, harrison Gomes Magdinier4,
Philip Noel Suffys4, Nalin Rastogi5, David
Couvin5, Rafael Silva Duarte6, karla Valéria
Batista Lima7
1
evandro chagas institute Pará, institut de
biologie intégrative de la cellule, Federal
university of rio de Janeiro
2
evandro chagas institute
3
state university of Pará
4
oswald cruz Foundation
5
Pasteur institute
6
Federal university of rio de Janeiro
7
evandro chagas institute, state university of
Pará
MISCLASSIFICATION BY SPOLIGOTYPING:
MuLTI-DRuG RESISTANT MYCOBACTERIuM
TuBERCuLOSIS OF ThE LATIN AMERICAN
MEDITERANEAN LINEAGE WRONGLY
IDENTIFIED AS MYCOBACTERIuM PINNIPEDII
(ST863)
Elis R. Dalla Costa1*, Sidra E. G. Vasconcelos2*,
Leonardo Esteves3, harrison M. Gomes3, Lia
L. Gomes Fiocruz3, Pedro E. A. Silva4, joão
Perdigão5, Miguel Viveiros5, Gisela unis6, Nalin
Rastogi7, Maria Lucia R. Rossetti3, Philip Noel
Suffys3
1
Fundação estadual de Produção e Pesquisa
em saúde – Fepps, Porto Alegre 2laboratório de
biologia Molecular Aplicada A Microbactérias,
instituto oswaldo cruz, Fiocruz, rio de Janeiro
3
Fundação estadual de Produção e Pesquisa
em saúde – Fepps, Porto Alegre; universidade
luterana Do brasil (ulbra/rs)
4
universidade Federal Do rio Grande, rio
Grande Do sul
5
centro de Patogénese Molecular, uria,
Faculdade de Farmácia Da universidade de
lisboa
6
Hospital sanatório Partenon (Hsp), Porto Alegre
7
Who supranational tb reference laboratory,
institut Pasteur de la Guadeloupe, Abymes,
Guadeloupe
*contributed equally
We recently detected one of the spoligotypes, SIt863,
characteristic for Mycobacterium pinnipedii, a species
of the Mycobacterium tuberculosis Complex (MtC), in
sputum sample from 10 pulmonary tuberculosis cases
residents of Porto Alegre, South Brazil. Because this
species is rarely encountered in humans, we further
COMPARISON OF IS6110, MIRu-VNTR AND
SPOLIGOTYPING METhODS WhICh ARE
uSED FOR ASSOCIATING OF PhYLOGENETIC
RELATIONS OF MuLTI DRuG RESISTANT (MDR)
MYCOBACTERIuM TuBERCuLOSIS ISOLATES
Gulfer Yakici1, Begum kayar2, Taylan Bozok1,
Togrul Nagiyev1, Mahdi Marzi1, Emel Yarar1, Fatih
koksal1
1
2
High incidence of tuberculosis in developed and
developing countries and appearing of strains with
resistance to two major drugs (multi drug resistant) in
addition to two minor drugs (extensively drug resistant)
and tendency to spread of beijing strains which do
not response to anti-tuberculosis treatment in all
around the world are the most important handicaps of
tuberculosis control programmes. This global problem
can be exceed by monitoring M. tuberculosis (MtB)
strains with more sensitive and more discriminative
molecular epidemiologic methods.
In this study we aimed to find contribution of combining
three methods which have high discriminative power
for epidemiologic studies in addition to research
relation between MDR-tB strains with IS6110-RFLP,
15 loci MIRu-vNtR and spoligotyping. We detected
110 MDR-tB strains from 22.619 clinical metarials
which were sent to Tropical Disease Center Region
tuberculosis Laboratory between june 2011- May
tuberculosis (tB) is an infectious disease that is
caused by Mycobacterium tuberculosis (MtB).
Although highly effective treatment schemes have
been available for decades, tB remains a major
global health problem, especially in the 22 countries
given high priority due to their high load of TB cases
(High-Burden Countries - HBCs) since 2000. Brazil
was ranked 17th among these in 2013. Molecular
typing of strains of MtB has improved understanding
of TB epidemiology and opened new avenues for
study - ones where transmission dynamics meet
with classical epidemiological approaches. One
technique, spoligotyping, is an especially useful tool
for the classification of strains of MtB. By performing
spoligotyping on a large number of MtB clinical
isolates, this study aimed to determine the major
87
characterized these isolates by additional genotyping
including 24 MIRu-vNtR typing, verification of tbD1,
RD9 and RDRio status, sequencing of pks15/1, insertion
of IS6110 at a specific locus for M. tuberculosis
lineage (Latin American Mediterranean/LAM) and the
presence of the SNP fbpC103. Combined analysis of
all these markers revealed that the isolates are in fact
a M. tuberculosis (LAM) and this was confirmed by
whole genome sequencing of three of the isolates and
“bar code” based identification. Most of these isolates
showed to be multi-drug resistant so were submitted
to partial sequencing of genes rpoA, rpoB, rpoC, katG
and inhA. Eight isolates (80%) showed the presence
of the AGC-ACC mutation at codon 315 of katG (Serthr) and one isolate (10%) exhibited a substitution
of T-G at -17 position of inhA. Interestingly, eight of
the MDR isolates also presented an undescribed
insertion of 12 nucleotides (CCA GAA CAA CCC)
at codon 516 of rpoB. No compensatory mutations
were observed in the genes rpoA and rpoC. This is
the first report on M. tuberculosis LAM family with a
convergent spoligotype characteristic for M. pinnipedii.
Because the M. pinnipedi spoligotypes are observed
in spoligotype databases with a certain frequency,
care must be taken when concluding on the identity of
isolates of the M. tuberculosis complex (MtC) based
on a single genetic marker.
P212
IDENTIFICATION
OF
NON-TuBERCuLOuS
MYCOBACTERIA
SPECIES
IN
ROuTINE
CLINICAL PRACTICE uSING MALDI-TOF MASS
SPECTROMETRY
Eva Sodja1, Nada Šorli Peranović1, Helena Ribič2,
Manca Žolnir-Dovč1
1
laboratory for Mycobacteria, university clinic
of respiratory and Allergic Diseases Golnik
2
environment and Food; centre for Medical
Microbiology, National laboratory of Health
Background: Significant number of new mycobacteria
species is described each year. therefore, there is an
urgent need for new methodologies to rapidly identify
clinically relevant non-tuberculous mycobacteria
(NtM) species isolated from clinical samples.
Matrix-assisted laser desorption time-of-flight mass
spectrometry (MALDI-toF MS) has been recently
proven to effectively identify mycobacteria directly
from cultures. The aim of our study was to compare
MALDI-toF MS Biotyper system (Bruker Daltonics,
Bremen, Germany) to Geno type Mycobaterium CM/
AS (GtM; Hain Lifescience, Nehren, Germany) and
to evaluate its use in our routine laboratory practice.
Methods: A total of 34 NtM isolates from various
clinical materials were tested in this study. NtM
subcultures grown on solid media (Middlebrook
7H10 or Löwenstein-jensen) were initially identified
with GtM assays. For MALDI-toF MS analysis,
the NtM colonies were inactivated by heating up
88
to 100°C and 0.5mm zirconia/silicate beads were
used for mechanical disruption, as recommended
by Bruker Daltonics. Protein extraction was elicited
with acetonitrile and fromic acid. The samples were
pipetted onto MALDI target plate and identified with
Bruker MALDI Biotyper using automatic spectral
acquisition and Bruker Mycobacteria Library 1.0.
Results: All mycobacterial isolates were identified
using GtM as NtM of which 5 were M. avium, 5 M.
chelone, 5 M. xenopi, 4 M. gordonae, 4 M. fortuitum, 2
M. intracellulare, 1 M. interjectum, 2 M. kansasii, 4 M.
abscessus and 2 M. fortuitum 2/M. mageritense. The
Bruker mycobacteria library gave a spectral scores
higher than 1.7 for 30 (88.2%) NtM isolates and 28
(93.3%) of these isolates were correctly identified to
the species level when compared with the GtM. the
remaining 4 (11.8%) NtM isolates yielded in spectral
scores lower than 1.7 (3) or no spectra were found
(1) during MALDI toF analysis. two (5.9%) isolates
identified as M. fortuitum2/M. mageritense with GtM
were identified as M. senegalense using mycobacteria
library (scores higher than 2.0) and will be subjected
to 16S rRNA sequencing.
Conclusion: According to our data, MADLI-toF
MS has potential for identifying mycobacteria in the
clinical laboratory practice, by reducing identification
turnaround time and laboratory costs. Nevertheless,
further work to optimize limiting factors (type of
medium used for mycobacteria growth, quantity
and age of the mycobacteria culture, maycobacteria
library with higher number of species) is still crucial.
P213
DRuG SuSCEPTIBILITY PROFILE AND
MOLECuLAR TYPING OF PREDOMINANT
SINGLE NuCLEOTIDE POLYMORPhISM (SNP)
CLuSTER GROuPS IN DELhI
Mandira Varma-Basil1, kushal Garima1, Anshika
Narang1, Soumitesh Chakravorty2, Naresh
kumar1, Shraddha Podwal1, Astha Giri1, Thierry
Zozio3, David Couvin3, Stefan Niemann4, Nalin
Rastogi3, David Alland2, Mridula Bose1
1
Vallabhbhai Patel chest institute, Dept. of
Microbiology, university of Delhi
2
Department of Medicine, New Jersey Medical
school, rutgers university
3
tuberculosis and Mycobacteria unit; Who
supranational tuberculosis reference
laboratory, institut Pasteur de la Guadeloupe
4
borstel, Germany
Phylogenetic analysis of M. tuberculosis populations
reveals associations between lineages that may
have implications in determining the effectiveness of
Tuberculosis control programs.
Several attempts have been made to associate the
differences in strains of M. tuberculosis to variations
in the clinical outcome of the disease and to drug
resistance. We genotyped 139 clinical isolates of
M. tuberculosis obtained from patients of pulmonary
tuberculosis and attending a referral center for
patients with respiratory diseases in Delhi, India. the
isolates were analyzed using nine Single Nucleotide
Polymorphism (SNP) markers. the isolates were also
subjected to spoligotyping and MIRu-vNtR typing;
and the results correlated with their drug susceptibility
profile.
The most predominant cluster was SNP cluster group
(SCG) 3a observed in 56% (n=78) of the isolates.
Majority (50%) of the isolates in SCG 3a were drug
susceptible, while 9% (n=7) were multidrug resistant
(MDR). the ancestral cluster SCG 1 was observed in
18% (n=25) of the isolates. Multidrug resistance was
observed in 8% (n=2) of these isolates while 40%
(n=10) of the isolates were drug susceptible. SCG
2 formed 6.2% (n=9) of the isolates and 44% (n=4)
of these were MDR. Spoligotyping subdivided the
strains into 45 shared types (n=125) and 14 orphan
strains. The orphan strains were mostly associated
with SCG 3a or SCG 1, reflecting the principal SCGs
found in the Indian population. SCG 1 and SCG 2
spoligotypes were concordant with the East African
Indian and Beijing families respectively. Central Asian
(CAS) clade and its sublineages were predominantly
associated with SCG 3a, however a few strains (n=3)
were also observed to be SCG 3b. No consistent
association was seen between the SCGs and other
spoligotypes such as Harlem, t and x clades.
Surprisingly, the 15 loci MIRu-vNtR typing revealed
all isolates to be unique.
In conclusion, though our study revealed the
preponderance of SCG 1 and 3a in the M. tuberculosis
population circulating the region, the diversity of
strains highlights the changes occurring within
lineages and reemphasizes the importance of cluster
investigations.
P219
STuDYING ThE METABOLIC ACTIVITY OF
MYCOBACTERIuM TuBERCuLOSIS ThROuGh
A NEW RESAZuRIN REDuCTION BASED ASSAY
Carla Silva1, joao Perdigao2, Luísa jordão3,
Isabel Portugal1
1
imed.ulisboa – instituto de investigação
Do Medicamento, Faculdade de Farmácia,
universidade de lisboa
2
uria, centro de Patogénese Molecular,
lisboa, Portugal
3
Departamento de Doenças infeciosas, instituto
Nacional de saúde Dr. ricardo Jorge
Lately, several approaches have been performed
to study the biological aspects of the mycobacterial
metabolism and elucidate some bacterial strategies
to survive to host responses, but little is known about
mycobacterial adaptation to adverse environmental
conditions. In this study we developed a simple
and rapid method to evaluate the metabolic rate of
Mycobacterium tuberculosis (M. tuberculosis) strains
in liquid culture media, based on the resazurin
reduction kinetics.
We tested four mycobacterial growth conditions:
physiological (pH 6.7), acidic (pH 5.5 and pH 4.5)
and anaerobic; the avirulent strain M. tuberculosis
H37Ra was cultured until log phase, and 3 different
bacterial concentrations (108, 106 and 105 CFu/ml)
were inoculated in triplicate, and incubated at 37ºC
with 5% Co2 atmosphere. After inoculation, 20µl
of resazurin 0,01% were added, and the indicated
optical density was measured at several time-points
(0h, 6h and 24h). this procedure was repeated three
days after the inoculation, concluding a 5 day period
of incubation. CFu counting was performed at the
time of inoculation, and at the end of experience for
each condition.
Globally, our results show an increasing resazurin
reduction curve dependent on the normal growth
curve of viable bacteria. However, after a three
day incubation period the percentage of resazurin
reduction kinetics was shown to be linear only for
cultures inoculated with 106 CFu/ml. this pattern was
also observed under acidic and anaerobic conditions.
The method was found to be reproducible with low
standard deviations. thus, we selected the 106 CFu/
ml as the best bacterial inoculum concentration for
this assay. Afterwards, we analysed the growth curve
of 106 CFu/ml culture for each conditions, and CFu
counting as well. Although the H37Ra strain has
the capability to grow in all conditions tested, which
was confirmed by CFu counting, it was possible to
distinguish the rate of resazurin reduction across
the different environments. As expected, a smaller
number of CFu/ml was obtained in acidic conditions
when compared to anaerobic and aerobic conditions.
taking into account these results, we believe that
the methodology here presented holds potential for
mycobacterial metabolic studies concerning different
strain physiology.
P220
IDENTIFICATION OF ThE BEST MIRu-VNTR
SET TEChNIquE FOR MDR-TB AND xDR-TB
SuRVEILLANCE IN LISBON, PORTuGAL
Carla Silva1, joao Perdigao2, Luisa jordao3,
Isabel Portugal1
1
2
lisboa, Portugal
3
Departamento de Doenças infeciosas, instituto
Nacional de saúde Dr. ricardo Jorge
89
Multidrug and extensively drug resistance tuberculosis
(MDR/xDR-tB) remains a serious threat to tB
control worldwide. Portugal registers a high number
of MDR-tB and xDR-tB cases particularly in Lisbon,
the capital city. the majority of MDR- and xDR-tB
cases that circulates in Lisbon belongs to either one
of two groups of genetically close strains, known
as Lisboa family and q1 cluster. these strains are
responsible for more than 80% of MDR-tB cases;
50% of which are also xDR-tB (2008). Given the
high prevalence and considering the drug resistant
profile of such strains, we aimed to investigate the
most discriminatory technique for MDR- and xDR-tB
surveillance.
For this purpose, 74 clinical Mycobacterium
tuberculosis isolates collected in Lisbon Health Region
were genotyped by 12, 15 and 24-loci Mycobacterial
interspersed repetitive units – variable number of
tandem repeats (MIRu-vNtR). twenty-two isolates
were susceptible and 54 were resistant to at least one
drug (34 MDR-tB, 15 of which were xDR-tB). We
verified that MIRu-vNtR analysis based on 12, 15
and 24 loci sets allowed the clustering of 22 (66,7%),
20 (60,6%) and 17 (51,5%) MDR-tB isolates; 13
(92,9%), 12 (85,7%) and 11 (78,6%) of these were
also xDR-tB, respectively. It was also noticed
that, as the number of analysed loci increased, the
Lisboa3 and q1 isolates that differ by one or two loci
were gradually excluded from the clusters. In order
to evaluate the clustering of MDR-tB and xDR-tB
isolates, we analysed the discriminatory power of all
MIRu-vNtR sets, applying the Simpson’s Diversity
Index. As expected, the 24 loci set analysis presented
the highest discriminatory index (D) in comparison
with both 12 and 15 loci sets (DMIRu 24=0,971; DMIRu
=0,964; DMIRu 12=0,931). the small difference
15
observed between DMIRu 15 and DMIRu 24, leads to the
conclusion that 15 loci MIRu-vNtR is a powerful
discriminatory tool. Interestingly, although with a
lower discriminatory power, the 12-loci MIRu-vNtR
analysis was enough to cluster the xDR-tB isolates
into two major clusters, Lisboa3 and q1 cluster,
corresponding to 92,9% of xDR-tB cases in Lisbon
Health Region. Despite having a lower discriminatory
power, we conclude that, in such settings, 15 loci
MIRu-vNtR has a sufficient discriminatory power
for MDR-tB and xDR-tB surveillance and that 12
loci MIRu-vNtR is a useful method to ascertain a
common origin between drug resistant isolates that
have undergone subsequent genetic diversification.
P221
DRuG RESISTANCE AND GENETIC DIVERSITY OF
MYCOBACTERIuM TuBERCuLOSIS IN LuANDA,
ANGOLA: A MOLECuLAR EPIDEMIOLOGICAL
PERSPECTIVE
joao Perdigao1, Sofia Clemente2, jorge Ramos3,
Pedro Masakidi2, Diana Machado3, Carla Silva4,
Isabel Couto3, Miguel Viveiros3, Nuno Taveira5,
90
Isabel Portugal4
1
lisboa, Portugal
2
serviço de Doenças infecciosas, Hospital Da
Divina Providência
3
Grupo de Micobactérias, unidade de
Microbiologia Médica, instituto de Higiene e
Medicina tropical de lisboa, universidade Nova
de lisboa
4
5
universidade de lisboa; centro de investigação
interdisciplinar egas Moniz, instituto superior
de ciências Da saúde egas Moniz, Monte de
caparica
tuberculosis (tB) poses a serious public health
problem in Angola. The latest estimates from the
World Health Organization point to the occurrence of
69 000 new cases in 2013 and an incidence rate of
320 cases per 100 000 habitants in the same period.
Furthermore, no surveillance data on drug resistance
is available and nothing is known regarding the
genetic diversity and population structure of circulating
Mycobacterium tuberculosis (M. tuberculosis) strains.
Its capital city, Luanda, harbours approximately 33.7%
of the country’s population and is responsible for onethird of the TB cases nationwide.
In the present study we have analysed 77 M.
tuberculosis isolates recovered from sputum samples
from the same number of patients. The patients
involved in the study were clinically diagnosed with
TB and positive for sputum smear acid fast bacilli in
Hospital da Divina Providência, Luanda. All isolates
were genotyped by Spoligotyping and 24-loci
Mycobacterial Interspersed Repetitive unit – variable
Number of tandem Repeats (MIRu-vNtR). Firstline drug susceptibility testing was performed by the
standard BACtEC 960 MGIt procedure.
We have detected 29 different spoligotype profiles
corresponding to 21 different Shared International
types (SIt) and 8 orphan profiles. SIt 42 (Latin
American Mediterranean, LAM9) was the most
prevalent SIt found (n=15, 19.5%) followed by SIt
53 (t1; n=11, 14.3%). overall, the M. tuberculosis
population structure in this sample was dominated
by LAM (57.1%) and t (32.5%) strains. twelve and
24-loci MIRu-vNtR analysis revealed that 47 (15
clusters) and 11 (4 clusters) isolates were clustered,
respectively.
Drug susceptibility data showed that 17 (21.1%) of
the 77 clinical isolates were resistant to one or more
antibacillary drugs and from these, 3 (3.9%) were
multidrug resistant (MDR). Drug resistant isolates
were found across distinct clades and MIRu-vNtR
clusters although the largest MIRu-vNtR cluster
detected was comprised by 3/5 drug resistant isolates,
including one MDR isolate
In conclusion, this study has demonstrated a high
predominance of LAM strains circulating in Luanda
and the presence of recent transmission events. The
genetic diversity found suggests the presence of
multiple and complex TB transmission chains masked
by recent genetic diversification. the high rate of
MDR-tB found in this sample has major public health
implications and highlights the need of further studies
specifically focused on MDR-tB transmission.
P223
that were shared with other Manila family clusters.
one of these clusters had five cases, composed of
four Micronesians and one uS born case, while the
other cluster had six cases including two cases born
in the Marshall Islands, two Filipinos, one Micronesian
and a uS case.
Periodic evaluation of genotyping information
has revealed possible transmission clusters and
highlighted the diversity of tuberculosis cases in
Hawaii. Linking genotypes to case data to determine
potential epidemiologic links is essential as we move
into whole genome sequencing of representatives of
these clusters, currently in progress.
CLuSTER IDENTIFICATION AND ANALYSIS OF
CLINICAL MYCOBACTERIuM TuBERCuLOSIS
ISOLATES IN ThE STATE OF hAWAII
kent koster, Lishi qian, Caitlin Flores, Terry
Weber, Richard Brostrom, jeffrey Foster, james
Douglas
university of Hawaii at Manoa, Honolulu, Hi
california state Dept. of Health, richmond, cA
Hawaii state Dept. of Health, Honolulu, Hi
university of New Hampshire, Durham, NH
Hawaii’s tuberculosis incidence rates are perennially
among the highest in the united States. Immigration
from the Western Pacific and Asia over the last
decade continues to introduce a steady supply of new
tuberculosis cases into the state, an average of 121
cases per year with an increase to over 136 cases
this past year. This is in contrast to the declining
number of cases in the uS over the same period.
As a result of this importation, distinguishing locally
transmitted tuberculosis cases from cases brought
into the state has been difficult when using traditional
epidemiological methods.
In this study, we analyzed data covering 439 isolates
collected by the Hawaii State Department of Health
from 2004 to 2013 to identify potential transmission
clusters. Genetic clusters were identified using
spoligotyping and MIRu-vNtR fingerprinting
methods.
Out of twenty genetic clusters consisting of three or
more isolates, we identified six major fully fingerprinted
clusters that included cases at least three years apart.
of these, two clusters were composed of isolates from
the Beijing family and four clusters from the Manila
family. one Beijing family cluster was composed of
seven isolates and consisted primarily of uS-born
Micronesian males age 15-24, while the other had
eleven isolates and consisted primarily of young to
middle-aged people of Pacific Islander descent, but
also included two North Korean-born women. Of the
largest Manila family clusters, one had 23 isolates
with six similar spoligotype patterns, while the other
had 24 isolates with three similar spoligotype patterns.
Both larger Manila family clusters were composed of
persons born in the Philippines and covered a broad
age range, with the 45-64 and 65+ age ranges being
most common. two more Manila family clusters were
tentatively identified by their MIRu loci 1-12 patterns
91
TOWARDS TB ELIMINATION
P127
ANALYTICAL SENSITIVITY (LIMIT OF
DETECTION), ANALYTICAL REACTIVITY
(INCLuSIVITY) AND ANALYTICAL SPECIFICITY
(CROSS-REACTIVITY) OF ThE BD MAx
MDR-TB ASSAY FOR ThE DETECTION OF
AND RIFAMPICIN AND ISONIAZID RESISTANCE
Ira Ashman1, Eliza Alexander1, Ashley Anderson1,
kalola Andrews1, kelley Bryan-McNeal1, krystal
hamlet1, jackie harris1, jeffry Leitch1, Stephanie
Suit1, Michael Porter1
1
becton Dickinson
sensitivity of the assay was found to be less than 50
cfu/mL-sputa for M. tuberculosis detection and less
than 250 cfu/mL-sputa for rifampicin and isoniazid
resistance detection. The assay successfully detected
all five M. tuberculosis complex organisms tested.
There was no detectable cross-reactivity with any of
the five NtMs.
the BD MAxtM MDR-tB assay is currently under
development and not available for sale or use.
Tuberculosis is a deadly infectious disease and
continues to remain one of the world’s top health
challenges. Rapid and accurate detection of
Mycobacterium tuberculosis and drug resistance is of
paramount importance to appropriately identify and
treat patients suffering with the disease, reduce the
death rate and stop the spread of TB.
the BD MAxtM MDR-tB assay is an automated,
qualitative test that allows for the direct detection of
M. tuberculosis complex and identification of genetic
mutations correlated with rifampicin (RIF) and isoniazid
(INH) resistance from raw (unprocessed) and NALC/
NaoH-treated (processed) clinical sputum samples.
the assay is designed for use on the BD MAxtM
System, which offers true walk-away automation by
incorporating sample lysis, extraction, amplification
and detection all in one system.
Analytical sensitivity of the assay for both M. tuberculosis
complex detection and mutant discrimination was
determined using spiked negative sputa. BD MAxtM
MDR-tB analytical sensitivity for the detection of
M. tuberculosis complex was evaluated using the
H37Rv strain. Mutant discrimination was assessed
using five clinical isolates. Mutant genotypes were
determined by Sanger sequencing and drug-resistant
phenotypes were determined by BACtEC MGIt 960
culture. Analytical sensitivity for the identification of a
RIF mono-resist strain was evaluated using a clinical
isolate with a His526Tyr substitution in rpob, while
analytical sensitivity for the identification of an INH
mono-resistant strain was evaluated using a clinical
isolate with a Ser315Thr substitution in katG. Analytical
sensitivity for detection of multi-drug resistant strains
was evaluated using three isolates with mutations
in both rpob and katG genes; namely, Ser531Leu/
Ser315thr, His526Asp/Ser315thr and Gln513Lys/
Ser315 rpob/katG mutants. Analytical inclusivity,
specificity, and cross-reactivity were verified for
five M. tuberculosis complex organisms and five
nontuberculous mycobacteria (NtMs). Analytical
92
93
AUTHOR INDEX
Abbasi, Shahid 25, 85
Abdallah, Abdallah 25, 84
Abdurrahman, Saddiq T. 24, 81
Abid, Mohammed 9, 14, 34
Afsar, Ilhan 18, 21, 52, 66-67
Ağir, Ismail 22, 71
Agnieszka, Broda 25, 83
Agonafir, Mulualem 22, 24, 72, 81
Aguilar Ayala, Diana Angelica 11, 16, 20, 47, 62
Ait Benhassou, Hassan 9, 14, 34
Akgun, Sadık 22, 71
Aklilu, Abiy 21, 66
Akthar, Naeem 25, 85
Alabi, Abraham 10, 15, 41
Aleksandrova, Natalia 22, 72
Alexander, Eliza 27, 92
Alexandru, Sofia 10, 15, 39
Al-Hajoj Al-Nakhli, Sahal 24, 80
Alland, David 26, 88
Alvarez Figueroa, Maria 12, 17, 22, 50, 72
Alvarez-Maya, Ikuri 24, 78
Ameni, Gobena 18, 53
Amzazi, Saaïd 9, 14, 34
Anderson, Ashley 27, 92
Andrews, Kalola 27, 92
Andries, Koen 20, 62
Anthony, Richard 10-11, 15, 38-39
Anzola, JM 23, 76
Ares, Miguel Angel 23, 75, 76
Aseffa, Abraham 11, 16, 43
Ashman, Ira 27, 92
Aspindzelashvili, Rusudan 10, 15, 38
Atilgan, Adnan 19, 59
Augustynowicz Kopeć, Ewa 18, 24, 54, 80
Azé, Jérôme 25, 85
Bablishvili, Nino 10, 15, 25, 38, 85
Balabanova, Yanina 12, 17, 48
Baldan, Rossella 12, 17, 51
Barilo, Valentina 12, 17, 50
Baskin, Huseyin 21, 67
Batista Lima, Karla Valéria 26, 87
Battaglia, Simone 11, 16, 45
Baumane, Margarita 24, 79
Baumanis, Viesturs 10, 15, 24, 38, 79
Bauskenieks, Matiss 10, 15, 24, 38, 79
Beau de Rochars, Madsen 19, 59
Beckert, Patrick 9-10, 14-15, 36, 41
Belén Orandoni, Maria 20, 63
Beltrán, Magda 22, 69-70
Bengaard Andersen, Aase 25, 86
Benić, Miroslav Benić 20, 63
Benitez-Capistros, Ricardo 11, 16, 44
Bentaleb, El Mehdi 9, 14, 34
Berg, Stefan 11, 16, 43
Bergval, Indra 10, 15, 38
94
Berland, Jean-Luc 25, 85
Bidault, Floriane 25, 85
Biyé Ondó, Lucía 9, 14, 36
Bjorn-Mortensen, Karen 25, 86
Blanco, Silvia 21-22, 24, 66, 72, 81
Blom, Jochen 21, 64
Bobrikova, Olga 24, 79
Boehme, Catharina 9, 13, 30
Bonnet, Maryline 10, 15, 41
Bonura, Celestino 11, 16, 43
Bordi, Eugenio 21, 68
Borek, Anna 24, 80
Borkowska, Dagmara 24, 80
Borrell, Sonia 30
Borroni, Emanuele 9, 11, 14, 16, 25, 35, 45, 83
Bose, Mridula 19, 26, 59, 88
Bouza,Emilio 9, 14, 25, 36, 82
Bozok, Taylan 19, 21, 26, 57, 59, 65, 86
Brandao, Angela 18, 53
Bruske, Ellen 10, 15, 41
Bryan-McNeal, Kelley 27, 92
Brzezińska, Sylwia 18, 54
Bull, Tim 22, 68
Bustos, JR 23, 76
Butera , Ornella 21, 68
Cabibbe, Andrea Maurizio 9, 11, 14, 16, 35, 36, 45
Cambau, Emmanuelle 8
Campana, Silvia 12, 17, 51
Cariani, Lisa 12, 17, 51
Carmen Menendez, Maria 23, 76
Castañeda-Casimiro, Jessica 23, 73
Castellvi, Josep 9, 14, 34
Catanzaro, Antonino 10, 15, 29, 39
Ceyhan, Ismail 23, 74
Chaburte, Chala 21, 66
Chakravorty, Soumitesh 26, 88
Chesov, Dumitru 10, 15, 39
Christianson, Sara 18, 52
Christoffels, Alan 25, 84
Cirillo, Daniela Maria 5, 8-9, 11, 12-14, 16, 17, 21, 25, 28,
35-36, 45, 51, 67, 83
Clemente, Sofia 26, 90
Cnockaert, Margo 20, 63
Coeck, Nele 19, 56, 58
Comas, Iñaki 9, 11, 14, 16, 36, 43
Conceição, Emilyn 26, 87
Couppié, Pierre 12, 17, 51
Couto, Isabel
26, 90
Couvin, David 12, 17, 26, 51, 87-88
Cox, Helen 30
Crudu, Valeriu 10, 15, 29, 39
Cuevas, Luis 22, 24, 72, 81
Cvetnić, Luka 20, 63
Cvetnić, Željko 20, 63
Cynamon, Michael 20, 61
Dalla Costa, Elis R. 26, 87
Datiko, Daniel 22, 24, 72, 81
David, Suzana 12
De Beer, Jessica 10, 15, 16, 45, 38
De Coninck, Dieter 11, 16, 47
De Haas, P. 16, 45
De Jong, Bouke C. 10, 15, 19, 41, 56, 58
De Keijzer, Jeroen 11, 16, 45
De Ru, A. 16, 45
De Viedma, Darío García 9, 12, 14, 25, 36, 82
De Vos, Margaretha 25, 84
Dedkov, Vladimir 22, 72
Deforce, Dieter 11, 16, 47
Del Portillo, P 9, 23, 76
Demirci, Mustafa 18, 52
Den Hertog, Alice 10, 15, 39
Di Caro, Antonino 21, 68
Dippenaar, Anzaan 25, 84
Dogonadze, Marine 23, 76
Dolinger, David 21, 67
Domente, Liliana 10, 15, 39
Dominguez, Jose 19, 22, 24, 56, 72, 81
Drewe, Julian 25, 84
Drobniewski, Francis 12, 17, 25, 48, 83
Dufera, Boja 21, 66
Duvnjak, Sanja 20, 63
Dyakova, Marina 23, 75
Eckelt, Elke 12, 17, 48
Engelthaler, Dave 8, 13
Erdoğdu, H.İbrahim 22, 71
Ertürk, Şükrü Mehmet 22, 71
Esmedlyaeva, Diljara 23, 75
Esteves, Leonardo 26, 87
Estrada Parra, Sergio 23, 73
Estrada-García, Iris 23, 73
Eyene, Juan 9, 14, 36
Fabio, Anna 18, 54
Felder, Marius 20, 62
Feleke, Abenezer 21, 66
Fellenberg, Kurt 9, 14, 36
Feres Saad, Maria Helena 19, 56
Ferrazoli, Lucilaine 18, 53
Feuerriegel, Silke 9, 14, 30, 36
Fitzgibbon, Margaret 23, 73
Forbicini, Giulia 18, 54
Francisco-Cruz, Alejandro 23, 73
Frank, Matthias 10, 15, 41
Frascaro, Francesca 18, 54
Fregni Serpini, Giulia 18, 54
Gagneux, Sebastien 10-11, 13, 16, 30-31, 43
Gao, Zhiwei 18, 52
Garcia, Maria Jesus 20, 23, 61, 76
Garfein, Richard 10, 15, 39
Garima, Kushal 26, 88
Gauthier, Marie 25, 85
Gehre, Florian 10, 15, 41
Gemechu, Abdella 18, 53
Gennari, William 18, 54
Gey van Pittius, Nico 25, 84
Gidaqy, Mirutse 18, 53
Giri, Astha 19, 26, 59, 88
Glader, Mikaela 11, 16, 46
Goethe, Ralph 12, 17, 48
Gomes, Meissiner 24, 81
Gomes Fiocruz, Lia L. 26, 87
Gomes Magdinier, Harrison 19, 26, 56, 87
Gomgnimbou, Michel 11, 16, 19, 22, 24, 43, 56, 72, 81
Gonzalez Perez, Monica Natalia 21, 64
Gonzalez y Merchand, Jorge 11, 16, 20, 23, 47, 62, 75,
76
Gozlan, Rodolphe 12, 17, 51
Groth, Marco 20, 62
Grottola, Antonella 18, 54
Guéguan, Jean-François 12, 17, 51
Guerra, Julio 19, 55
Gusarevica, Nina 24, 79
Hamdan, Azhar 22, 70
Hamlet, Krystal 27, 92
Harris, Jackie 27, 92
Heffernan, Courtney 18, 52
Helguera, C 23, 76
Hernandez, AC 23, 76
Hernández-Pando, Rogelio 23, 73
Herranz, Marta 9, 14, 25, 36, 82
Hillemann, Doris 9, 14, 35
Hilpert, Kai 22, 68
Hnilicová, Jarmila 12, 17, 20, 50, 61
Hoffner, Sven 5, 9, 11, 14, 16, 35, 46
Hölscher, Michael 10, 13, 15, 30, 41
Hölzer, Martin 20, 62
Ignatyeva, Olga 12, 17, 48
Igumnova, Viktorija 25, 83
Imperiale, Belen 18, 53
Iqbal, Riwan 25, 85
Izco, Santiago 9, 14, 36
Jang, Yoonra 24, 78
Jang, Yun-Ho 24, 78
Janoušková, Martina 12, 17, 20, 50, 61
Jansone, Inta 10, 15, 24-25, 38, 79, 83
Jarek, Michael 12, 17, 48
Johanna Pérez Llanos, Francy 22, 69-70
Johnson, Daniel 21, 67
Jordão, Luísa 26, 89
Karsli, Firat 19, 57
Katalinić-Janković, Vera 20, 60, 63
Kaya, Selcuk 18, 21, 52, 66-67
Kayar, Begum 19, 21, 26, 57, 59, 65, 86
Khan, Md. Siddiqur Rahman 19, 59
Kim, Jae Myung 24, 78
Kim, Jin Kyoung 24, 78
Kim, Jong-Taek 24, 78
Kim, Narae 24, 78
Klatt, Magdalena 24, 80
Klotoe, Bernice 19, 56
Knudson, Angélica 22, 69-70
Koch, Anders 25, 86
Kohansal, Farhad 19, 21, 57, 65
Kohl, Thomas A. 9, 10, 12, 14, 15, 17, 25, 30, 36, 41, 51,
86
95
Köhler, Heike 20, 62
Koksal, Fatih 19, 21, 26, 57, 59, 65, 86
Kompes, Gordan 20, 63
Kononenko, Julia 24, 79
Kontsevaya, Irina 12, 17, 48
Köser, Claudio 8, 13, 33
Kozińska, Monika 18, 24, 54, 80
Krásný, Libor 12, 17, 20, 50, 61
Kumar, Naresh 26, 88
Kumar, Parveen 18, 53
Ladefoged, Karin 25, 86
Lange, Christoph 10, 15, 39
Lauska, Zita 24, 79
Lauzardo, Michael 19, 59
Lawson, Lovett 24, 81
Lee, Hang 24, 78
Lee, Soo Hee 24, 78
Legrand, Eric 12, 17, 51
Leimane, Vaira 10, 15, 38
Leitch, Jeffry 27, 92
Lell, Bertrand 10, 15, 41
Levi, Diana 22, 72
Lillebaek, Troels 25, 86
Long, David 18, 52
Long, Richard 18, 52
Lopes, Maria Luiza 26, 87
Lopez, Beatriz 20, 63
López Roa, Paula 9, 14, 36
Lopez-Avalos, Gladys 24, 78
Lopez-Rodriguez, Martin 24, 78
Ludannyy, Ruslan 22, 72
Machado, Diana 26, 90
Magnani, Rita 18, 54
Mammina, Caterina 11, 16, 43
Manicheva, Olga 23, 76
Mansjö, Mikael 9, 14, 35
Marina, Maria Concepción 9, 14, 34
Markelov, Michail 22, 72
Martin, Anandi 10-11, 15-16, 18, 20, 42, 47, 53, 62-63
Martínez Lirola, Miguel 9, 14, 25, 36, 82
Marz, Manja 20, 62
Marzi, Mahdi 19, 26, 57, 86
Masakidi, Pedro 26, 90
Mata-Espinosa, Dulce 23, 73
May, R. Justin 19, 59
Mazzarelli, Antonio 21, 68
Meens, Jochen 12, 17, 48
Melnikova, Natalia 23-24, 76, 79
Merker, Matthias 9, 10, 13, 15, 30, 39
Millet, Julie 12, 17, 51
Miotto, Paolo
9, 11, 14, 16, 21, 35-36, 45, 67
Miralles, Pilar 25, 82
Möbius, Petra 20, 62
Mokrousov, Igor 10, 15, 23-24, 41, 76, 79
Molina-Moya, Barbara 19, 22, 24, 56, 72, 81
Momo, Juan Carlos 9, 14, 36
Monteserin, Johana 20, 63
Morcillo, Nora 18, 53
96
Morozova, Ilze 10, 15, 38
Morris, Aaron 12, 17, 51
Mosnier, Amandine 25, 85
Mougari, Faiza 8
Moure, Zaira 9, 14, 34
Mulder, A. 16, 45
Müllerová, Maria 22, 69
Munshi, Tulika 22, 68
Murcia, Martha I 18-19, 21-22, 55, 64, 69-71
Nagiyev, Togrul 19, 21, 26, 57, 65, 86
Nanni, Nadia 18, 54
Napiórkowska, Agnieszka 18, 54
Narang, Anshika 19, 26, 59, 88
Narvskaya, Olga 23-24, 76, 79
Navarrete, Myriam 22, 69-70
Navarro, Yurena 25, 82
Nicol, Marc 10, 13, 30
Niegowska, Magda 23, 77
Niemann, Stefan 2, 5, 8-10, 12-15, 17, 21, 23, 25-26, 28,
30, 36, 39, 41, 51, 67, 77, 86, 88
Nikolayevskyy, Vladyslav 12, 17, 25, 48, 83
Nisii, Carla 21, 68
Nodieva, Anda 10, 15, 24-25, 38, 79, 83
Nordsiek, Gabriele 20, 62
Noroc, Ecaterina 10, 15, 39
Ntoumi, Francine 10, 15, 41
Nübel, Ulrich 30
Obasanya, Joshua 24, 81
Obrovac, Mihaela 20, 60
Ocheretina, Oksana 25, 85
Ossa, Saray 22, 71
Ozere, Iveta 10, 15, 24-25, 38, 79, 83
Paboriboune, Phimpha 25, 85
Paccagnini, Daniela 23, 77
Paglia, Maria Grazia 21, 68
Pain, Arnab 25, 84
Palomino, Juan Carlos 10-11, 15-16, 18, 20, 42, 47, 53,
62, 63
Pape, Jean William 25, 85
Paranhos-Baccala, Glaucia 25, 85
Park, So-young 24, 78
Parra-López, Carlos 21, 22, 64, 69-70
Parsons, Sven 25, 84
Pastare, Ruta 24, 79
Pate, Mateja 20, 63
Paul, Roxana 20, 63
Pecorari, Monica 18, 54
Penlap Beng, Véronique N. 10, 15, 41
Peranović, Nada Šorli 26, 88
Perdigão, João 26, 87, 89-90
Pérez-Lago, Laura 9, 14, 25, 36, 82
Perini Furlaneto, Ismari 26, 87
Petik, Bülent 22, 71
Petrou, Matthieu 12, 17, 47
Pimkina, Edita 12, 17, 48
Pino, Camilo 18, 55
Piscini, Alessandro 21, 68
Pjalkovskis, Janis 25, 83
Platzer, Matthias 20, 62
Podwal, Shraddha 26, 88
Pole, Ilva 10, 15, 24-25, 38, 79, 83
Polo, Claudia Llerena 19, 55
Portaels, Francoise 11, 16, 44
Porter, Michael 27, 92
Portugal, Isabel 26, 89-90
Pospíšil, Jiří 12, 17, 20, 50, 61
Proano-Perez, Freddy 11, 16, 44
Prokopenko, Anastasiya 12, 17, 50
Rabodoarivelo, Marie Sylvianne 10, 15, 18, 42, 53
Rachow, Andrea 10, 15, 41
Raftery, Philomena 23, 73
Rakotomanana, Fanjasoa 10, 15, 42
Ramos, Jorge 26, 90
Rancoita, Paola Maria 12, 17, 51
Ranka, Renate 10, 15, 24-25, 38, 79, 83
Rasolofo, Voahangy 10, 15, 18, 42, 53
Rastogi, Nalin 5, 12, 17, 26, 51, 87-88
Ratovonirina, Noël 10, 15, 42
Razafimahatratra, Solohery 10, 15, 42
Refrégier, Guislaine 11-12, 16-17, 22, 24-25, 43, 47, 72,
81, 85
Reichwald, Katrin 20, 62
Reil, Irena 20, 63
Reynaud, Yann 12, 17, 51
Ribič, Helena 26, 88
Riekstina, Vija 25, 83
Rigouts, Leen 10-11, 16, 19, 44, 56, 58
Rimis, Constantin 10, 15, 39
Rios-Sarabia, Nora 23, 75
Ritacco, Viviana 20, 63
Rodríguez, Juan Germán 18-19, 22, 23, 55, 70-71, 76
Rodwell, Timothy 10, 15, 21, 39, 67
Romancenco, Elena 10, 15, 39
Ron-Garrido, Lenin 11, 16, 44
Rossetti, Maria Lucia R. 26, 87
Ruiz Serrano, María Jesús 9, 14, 25, 36, 82
Ruiz-Sánchez, Bibiana Patricia 23, 73
Rumpianesi, Fabio 18, 54
Rüsch-Gerdes, Sabine 30
Ryoo, Soyoon 24, 78
Saglam, Yusuf 18, 21, 52, 67
Sajid, Ali 19, 56
Sampson, Samantha 25, 84
Sánchez, Adrian 9, 14, 34
Sánchez, Liliana 22, 69-70
Sánchez, Ricardo 22, 69-70
Sanchez-Padilla, Elisabeth 10, 15, 41
Sandoval-Diaz, Manuel 24, 78
Sari, Abdülkadir 22
Sayiner, Hakan 22, 71
Schats, Mirjam 19, 58
Schena, Elisa 11, 16, 45
Schito, Marco 21, 67
Schleusener, Viola 9, 14, 36
Sechi, Leonardo A 23, 77
Sefrioui, Hassan 9, 14, 34
Sener, Aslı Gamze 21, 66-67
Sengstake, Sarah 10, 15, 38-39
Serafín López, Jeanet 23, 73
Sertel Şelale, Deniz 9, 14, 37
Sharma, Meenu 18, 52
Shipulin, German 22, 72
Shoen, Carolyn 20, 61
Siame, Keith 25, 84
Siddiqi, Salman 8
Šiková, Michaela 12, 17, 50
Silva, Carla 26, 89-90
Silva, Pedro E. A. 26, 87
Silva Duarte, Rafael 26, 87
Simonetti, Tullia 12, 17, 51
Singh, Sarman 18, 53
Skangale, Anita 24, 79
Skenders, Girts 8-10, 12, 15, 17, 24-25, 38, 48, 79, 83
Sklaney, Mary 20, 61
Skrahina, Alena M. 9, 14, 35
Sng, Li-Hwei 22, 70
Soborg, Bolette 25, 86
Sodja, Eva 26, 88
Soini, Hanna 9
Sola, Christophe 10-12, 15-17, 19, 22, 24-25, 42-43, 47,
56, 72, 81, 85
Solovieva, Natalia 24, 79
Soltan, Viorel 10, 15, 39
Somphavong, Silaphet 25, 85
Song, Woong-seog 24, 78
Spicic, Silvio 20, 63
Spinasse, Lizania 22, 24, 72, 81
Starkova, Daria 24, 79
Starks, Angela 21, 67
Stevens, Wendy 9, 13, 30
Stucki, David 11, 16, 43
Suffys, Philip Noel 26, 87
Suit, Stephanie 27, 92
Sunchalina, Tatiana 24, 79
Tabassum, Rubina 25, 85
Taccetti, Giovanni 12, 17, 51
Tagliani, Elisa 9, 14, 35
Tagliazucchi, Sara 18, 54
Tahseen, Sabira 25, 85
Tan, Yen Ee 22, 70
Tarhan, Gülnur 22, 71
Tauch, Andreas 18, 21, 55, 64
Taveira, Nuno 26, 90
Titarenko, Olga 23, 75
Tizzano, Barbara 23, 77
Toro, Juan Carlos 9, 14, 35
Tortola Fernandez, Maria Teresa 9, 14, 34
Tortoli, Enrico 12-13, 17, 32, 51
Trovato, Alberto 12, 17, 25, 51, 83
Tukvadze, Nestani 10, 15, 25, 38, 85
Unis, Gisela 26, 87
Uzun, Meltem 9, 14, 37
Van der Merwe, Ruben 25, 84
Van der Spuy , Gian 25, 84
97
Van Helden, Paul 25, 84
Van Nieuwerburgh, Filip 11, 16, 47
Van Soolingen, Dick 10, 15-16, 38, 45
Van Veelen, P. 16, 45
Vandamme, Peter 11, 16, 18, 20, 47, 53, 62-63
Varghese, Bright 24, 80
Varma-Basil, Mandira 19, 26, 59, 88
Vasconcelos, Sidra E. G. 26, 87
Vázquez-Chacon, Carlos 24, 78
Venditti, Carolina 21, 68
Villanueva-Arias, Juan Carlos 24, 78
Vishnevskiy, Boris 23, 76
Viveiros, Miguel 26, 87, 90
Vulcano, Antonella 21, 68
Vyazovaya, Anna 23-24, 76, 79
Walker, Timothy 8-9, 13, 29, 31
Warren, Rob 25, 84
Wasiak, Katarzyna 24, 80
Werngren, Jim 11, 16, 46
White, Richard 11, 13, 31
Wirth, Thierry 30
Woei Ling Peh, Justine 22, 70
Wolfe, Joyce 18, 52
Wong-Baeza, Isabel 23, 73
Worku, Adane 18, 53
Yakici, Gulfer 19, 21, 26, 57, 65, 86
Yarar, Emel 19, 21, 26, 57, 65, 86
Yasmin, Memona 25, 85
Young, Douglas 11, 16, 43
Yula, Erkan 21, 67
Zabost, Anna 18, 54
Zalutskaya, Aksana 9, 14, 35
Zambrano, MM 23, 76
Zdelar-Tuk, Maja 20, 63
Zhou, Hong 18, 52
Zhuravlev, Viatcheslav 23-24, 75-76, 79
Zignol, Matteo 21, 67
Zmak, Ljiljana 20, 60
Zmaznova, Anna 23, 76
Žolnir-Dovč, Manca 26, 88
Zozio, Thierry 26, 88
.
98

Scientific Program including Abstracts

Transcription

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