Florence 2013 - European Society of Mycobacteriology

Transcription

th
34
Annual Congress
of the European Society
of Mycobacteriology
30th June – 03rd July 2013
Florence, Italy
Scientific Program
including Abstracts
1
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Editor
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2
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CONTENT
Welcome Message ................................................ 4
Congress Organization ........................................ 5
Sponsors / Exhibition ........................................... 6
Scientific Program ................................................ 8
Invitation 2014 ..................................................... 13
List of Lectures ................................................... 14
Lectures (L) ..................................................................... 14
Guest Lectures (GL) ....................................................... 14
Honoree Lecture ............................................................. 15
Gertrud Meissner Award ................................................ 15
List of Oral Presentations (OP) ......................... 16
List of Poster Presentations (P) ........................ 20
Abstracts of Lectures ......................................... 33
Lectures (L) ..................................................................... 33
Guest Lectures (GL) ....................................................... 35
Honoree Lecture ............................................................. 40
Gertrud Meissner Award ................................................ 40
Abstracts of Oral Presentations ........................ 41
Abstracts of Poster Presentations .................... 59
Author Index ..................................................... 118
3
Dear Colleagues,
it is a pleasure to welcome you to Florence for the 34th annual meeting of the
European Society of Mycobacteriology. We are honored to have participants
coming from all over the world.
We hope that all of you will appreciate not only the great hallmarks of renaissance
butalsotheinformalandstimulatingscientificenvironmentthathasalways
characterized the ESM meetings.
Wewishyouafruitfulmeeting,excitingscientificexchangeandagreatmemory
to bring back home.
We are looking forward to meeting you in Florence.
Daniela Maria Cirillo
4
Enrico Tortoli
CONGRESS ORGANIZATION
SCIENTIFIC ORGANIZATION
Dr. Daniela Maria Cirillo
(Emerging Bacterial Pathogens Unit San Raffaele Scientific Institute, Milano)
Dr. Enrico Tortoli
(Emerging Bacterial Pathogens Unit San Raffaele Scientific Institute, Milano)
STEERING COMMITEE
President Stefan Niemann (Borstel, Germany)
Vice-president Gaby E. Pfyffer (Luzern, Switzerland)
Treasurer Nalin Rastogi (Pointe-A-Pitre, Guadeloupe, France)
Secretary Suzana David (Lisbon, Portugal)
Members Daniela Maria Cirillo (Milano, Italy)
Sven Hoffner (Solna, Sweden)
Girts Skenders (Spotinu Novads, Latvia)
LOCAL ORGANIZING AGENCY
Agency KONSENS GmbH
Germany
LOCAL CO-ORGANIZING AGENCY
The line Business
Luisa Chiesa
Italy
5
SPONSORS / ExHIBITION
We express our gratitude to all who supported and helped the organization of the 34th edition
of ESM congress:
Platin Medal Sponsor
Hain Lifescience GmbH
www.hain-lifescience.com
Booth number: 1
Gold Medal Sponsor
BD Diagnostics – Diagnostic Systems
www.bd.com
Booth number: 2
Other Sponsors
Cepheid Europe S.A.
www.cepheid.com
Booth number: 3
Salubris, Inc.
www.salubrisinc.com
Booth number: 4
Alpha-Tec Systems Inc.
www.alphatecsystems.com
Booth number: 5
Microsens Medtech Ltd.
www.microsens.co.uk
Booth number: 6
Bio-Rad Laboratories Ltd.
www.bio-rad.com
Booth number: 7
veredus Laboratories PTE Ltd.
www.vereduslabs.com
Booth number: 8
BioMérieux
www.biomerieux.com
Booth number: 9
Luminex B.v.
www.luminexcorp.com
Booth number: 10
Applied Maths Nv
www.applied-maths.com
Booth number: 11
Genoscreen
www.genoscreen.com
Booth number: 11
Alere Technologies GmbH
www.alere-technologies.com
6
7
SCIENTIFIC PROGRAM
Sunday, June 30, 2013
12:00-14:00 Registration at the Hotel Demidoff in Florence
14:00-16:00 Symposium: Genome based molecular epidemiology
Lecture 1: Towards genomic epidemiology of Mycobacterium tuberculosis, Philip Supply
Lecture 2: Dealing with uncertainties - interpreting genomic data for public health action,
Timothy Walker
Lecture 3: Advances in molecular typing of M. tuberculosis:
should WGS become the gold standard?, Dick van Soolingen
Lecture 4: Bench Top Next generation sequencing: Revolutionizing molecular epidemiology
and diagnostics?, Stefan Niemann
Round table discussion with experts
16:00-16:30 Coffee break in the foyer and visit of the exhibition
16:30-17:00 Symposium organized by HAIN
16:30-16:45
Molecular second-line drug testing - facts and needs
Doris Hillemann
16:45-17:00
Optimizing the Use of TB Molecular Diagnostics in Zambia
Barry Kosloff
17:00-17:20
Symposium organized by BD
First TB quantitative second line drug susceptibility testing study using TB eXist :
results and future developments
Emmanuelle Cambau
17:20-18:00
Opening session
Welcome
Memorial M. Magnusson and A. Arne
Malin Ridell
18:00-19:00 Honoree Lecture
Tuberculosis: new drugs on the horizon
Giovanna Riccardi (Italy)
19:00-19:30 Gertrud Meissner Award Lecture
Causes and consequences of parallel evolution of tuberculosis
and humans over 70.000 years
Inaki Comas
19:30
Transfer to “Castello di Vincigliata”
20:00
Welcome Reception at “Castello di Vincigliata”
23:00
Bus departure from “Castello di Vincigliata” back to all hotels
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Monday, July 01, 2013
09:00-10:30 M. TUBERCULOSIS GENOMICS I
Chairmen: Philip Supply, Dick van Soolingen
09:00-09:45
Guest Lecture 1: NEW INSIGHTS INTO PATHOGENOMICS OF THE TUBERCULOSIS AGENT
Roland Brosch (France)
09:45-10:00
Identification of lineage-specific genetic markers from more than
1,500 MTBC isolates (OP 39)
Francesc Coll, Kim Mallard, Mark D. Preston, Stephen Bentley, Julian Parkhill, Ruth McNerney,
Nigel Martin, Taane G Clark
10:00-10:15 Comparative transcriptomic analysis of Mycobacterium tuberculosis
in a lipid environment (OP 157)
Patricia Del Portillo, Maria Jesus Garcia, Jorge Gonzalez-y-Merchand, Maria Mercedes Zambrano,
Juan Germán Rodríguez, Juan Manuel Anzola
10:15-10:30 Insights into the gene activity of Mycobacterium tuberculosis growing
in a fatty-acid enriched medium (OP 53)
Juan German Rodriguez, Adriana Carolina Hernandez, Jorge Gonzalez-y-Merchand,
Addy Cecilia Helguera-Repetto, Jose Ricardo Bustos, Juan Manuel Anzola,
Maria Mercedes Zambrano, Patricia del Portillo, Maria Jesus Garcia
10:30-11:00
Coffee break in the foyer and visit of the exhibition
11:00-12:15
M. TUBERCULOSIS GENOMICS II
Chairmen: Sebastien Gagneux, Patricia Del Portillo
11:00-11:15 Generation and characterization of a M. tuberculosis deletion mutant
deficient in a putative subunit of a heterodimeric ABC multidrug
transporter (OP 246)
Sven Malm, Silvia Maaß, Stefan Ehlers, Stefan Niemann
11:15-11:30 Characterization of the small RNA expression profiles induced by
antibiotics in Mycobacterium tuberculosis (OP 164)
Paolo Miotto, Matthias Merker, Barbara Tizzano, Davide Cittaro, Dejan Lazarevic, Elia Stupka,
Stefan Niemann, Daniela M. Cirillo
11:30-11:45
Understanding the biology of poly-rifampicin resistance in Mycobacterium
tuberculosis (OP 23)
Margaretha de Vos, Gail Erika Louw, Rob Warren, Thomas Victor, Paul van Helden
11:45-12:00 Whole genome sequencing from early culture or directly from clinical
samples (OP 260)
Mathilde Mairey, Bénédicte Condamine, Christine Hubans-Pierlot, Stéphanie Ferreira,
Philip Supply, Caroline Allix-Béguec
12:00-12:15 Genome wide association analysis of tuberculosis resistance in
dairy cattle (OP 245)
Mairead Bermingham, Steve Bishop, John Woolliams, Adrian Allen, Stewart McBride,
David Wright, Jon Ryder, Robin Skuce, Stanley McDowell, Liz Glass
12:15-13:15 Poster session
13:15-14:00 Lunch
14:00-15:30 MOLECULAR EPIDEMIOLOGY OF TUBERCULOSIS AND STRATEGY FOR CONTROL
Chairmen: Nalin Rastogi , Kathleen Eisenach
14:00-14:45
Guest Lecture 2: MOLECULAR EPIDEMIOLOGY OF TUBERCULOSIS SPREADING
IN EUROPEAN METROPOLITAN AREAS
Andrea Gori (Italy)
9
14:45-15:00 Successful Russian clone of MYCOBACTERIUM tuberculosis:
origin, emergence and current spread (OP 47)
Igor Mokrousov
15:00-15:15 Large sequence polymorphism of Latin-American-Mediterranean (LAM)
Mycobacterium tuberculosis strains isolated in Italy and molecular
epidemiology of the LAM RDRio strains (OP73)
Laura Rindi, Nicola Bimbi, Carlo Garzelli
15:15-15:30 Tracing MYCOBACTERIUM tuberculosis transmission:
when resolution matters (OP 224)
Thomas Kohl, Roland Diel, Stefan Niemann
16:00-17:00 NDWG SYMPOSIUM
16:00-16:25
Guest Lecture 3: Rapid whole genome sequencing as a tool to manage
MDR/XDR-TB patients
Philip Butcher (UK)
16:25-16:50 Guest Lecture 4: LESSONS ON DRUG RESISTANCE FROM WGS
OF ONE THOUSAND CLINICAL ISOLATES
Taane Clark (UK)
16:50-17:15 Guest Lecture 5: Low density microarrays as potential tools for the
diagnosis of drug resistant tuberculosis
Paolo Miotto (Italy)
17:15-19:15 Sight Seeing Tour Florence
20:00
Traditional Dinner at “Convitto della Calza”
The tour guides will bring you directly from the city of Florence to the traditional restaurant
“Convitto della Calza”
23:00
Bus Departure from “Convitto della Calza” back to all hotels
Tuesday, July 02, 2013
09.00-10.45
MDR DIAGNOSTIC AND DST I
Chairmen: Daniela Maria Cirillo, Sabine Ruesch-Gerdes
09:00-09:45 Guest Lecture 6: Molecular and quantitative approaches to DST – tools
towards better guided TB therapy in the days of MDR and XDR
Erik Christian Boettger. (Switzerland)
09:45-10:00 Detection of rifampicin resistance in Mycobacterium tuberculosis by
padlock probes and a magnetic nanobead-based readout (OP 43)
Anna Engström, Teresa Zardán Gómez De La Torre, Maria Strømme, Mats Nilsson,
David Herthnek2
10:00-10:15
SPRINT: Spoligo-RifampICIn-Isoniazid Typing (OP 89)
Michel Gomgnimbou, Ivan Hernández-Neuta, Stefan Panaiotov, Elizabeta Bachiyska,
Juan Carlos Palomino, Anandi Martin, Patricia Del Portillo, Guislaine Refrégier, Christophe Sola
10:15-10:30 Development of an in vitro system to perform time-kill curves of
levofloxacin against Mycobacterium tuberculosis (OP 154)
Aline Barth, Robert May, Judith Johnson, Charles Peloquin, Hartmut Derendorf
10
10:30-10:45
Rapid testing for drug susceptibility (OP 212)
Antonino Catanzaro, Timothy Rodwell, Camilla Rodrigues, Valeriu Crudu, Thomas Victor,
Kanchan Ajbani, Elena Romancenco, Andre Trollip, Cindy Hayes, Roberta Lynn Jackson,
Theodore Ganiats, Erik Groessl, Richard Garfein
10:45-11:15
11:15-13:00
MDR DIAGNOSTIC AND DST II
Chairmen: Emmanuelle Cambau, Gabriela Pfyffer von Altishofen.
11:15-12:00
Guest Lecture 7: Challenges of pyrazinamide testing
Sven Hoffner (Sweden)
12:00-12:15
Kanamycin, amikacin, capreomycin cross-resistance in Mycobacterium
tuberculosis isolates from Romania (OP 229)
Daniela Homorodean, Andrea Melinda Jodal, Sven Hoffner
12:15-12:30 Epidemiological trends in TB and M/XDRTB and progress on the
implementation of the Consolidated Action Plan to Prevent and Combat
M/XDR-TB in the WHO European region (OP 243)
Kristin Kremer, Andrei Dadu, Martin van den Boom, Pierpaolo de Colombani, Masoud Dara
12:30-12:45 Emergence of XDR in high burden MDRTB country: a threat to public
health and TB control Program (OP 244)
Elena Romancenco, Valeriu Crudu, Ecaterina Noroc, Nadejda Turcanu, Liliana Domente,
Sofia Alexandru
12:45-13:00 Mycobacterium tuberculosis Beijing genotype strains are capable of
resisting transient exposure to rifampicin, as studied in a novel
microcolony approach (OP 96)
Alice den Hertog, Sandra Menting, Dick van Soolingen, Richard Anthony
13:00-13:45 Lunch
13:45-14:45 Poster session
14:45-16:15 New therapeutic regimens for TB and MDR-TB
Chairmen: Antonio Catanzaro, Vera Katalinic-Jankovic
14:45-15:30
Guest Lecture 8: New approaches to tuberculosis treatment
Christian Lienhardt (Switzerland)
15:30-15:45
Anti-tuberculosis activity of efflux inhibitors against drug resistant
Mycobacterium tuberculosis (OP 46)
Diana Machado, Isabel Couto, Jorge Ramos, Miguel Viveiros
15:45-16:00 Oral phenylbutyrate and vitamin D3 induce the cathelicidin LL-37 in immune
cells: A dose finding study for treatment of tuberculosis (OP 66)
Rubhana Raqib, Akhirunnesa Mily, Rokeya Rekha, S.M.Mostafa Kamal, Protim Sarker,
Zeaur Rahim, Gudmundur Gudmundsson, Birgitta Agerberth
16:00-16:15 Sterilizing activity of drug combinations against actively replicating and
nonreplicating Mycobacterium tuberculosis (OP 225)
Giovanni Piccaro, Federico Giannoni, Donatella Pietraforte, Alessandro Mustazzolu,
Lanfranco Fattorini
16:45-18:15 TB BIOMARKERS AND NOVEL VACCINE STRATEGIES
Chairmen: Maria Jesus Garcia, Stefan Niemann
11
16:45-17:30
Guest Lecture 9: Host-based biomarkers and different outcomes of
MTB infection
Gerhard Walzl (South Africa)
17:30-18:15
Guest Lecture 10: New TB vaccines, where we are and where we go
Carlos Martin (Spain)
18:15-19:00 General Assembly
20:30
Tuscan Barbecue in the garden and restaurant in Hotel Demidoff
Afterwards you are invited to the party in Hotel Demidoff.
Wednesday, July 03, 2013
09:00-10:45 NONTUBERCULOUS MYCOBACTERIA
Chairmen: Manuel J. Casal, Enrico Tortoli
9:00-09:45
Guest Lecture 11: New challenges for Buruli ulcer control
Françoise Portaels (Belgium)
09:45-10:00 What is the role of horizontal gene transfer in mycobacterial drug
resistance and virulence (OP 64)
Sylvia Cardoso Leão, Katiane Santin, Christiane Lourenco Nogueira, Paulo Jose Martins Bispo,
Cristianne Kayoko Matsumoto
10:00-10:15 Molecular characterization of Mycobacterium avium clinical strains
from St. Petersburg, Russia (OP 75)
Daria Starkova, Tatiana Otten, Igor Mokrousov, Anna Vyazovaya, Boris Vishnevskiy,
Olga Narvskaya
10:15-10:30
Whole genome sequencing provides evidence for transmission of
Mycobacterium abscessus between cystic fibrosis patients (OP 113)
Josephine Bryant, Dorothy Grogono, Daniel Greaves, Juliet Foweraker,
Iain Roddick, Thomas Inns, Mark Reacher, Charles S. Haworth,
Martin D. Curran, Simon R. Harris, Sharon J. Peacock, Julian Parkhill,
Andres Floto
10:30-10:45 Emergence of clinically relevant non-tuberculous mycobacterial
infections in Saudi Arabia.( OP 159)
Bright Varghese, Ziad Memish, Naela Abouljadayel, Raafat Al-Hakeem, Fahad Alrabiah,
Sahal Al-Hajoj Al-Nakhli
10:45-11:15
11:15-12:30
IGRA AND LATENT TB
Chairmen: Suzana David, Lanfranco Fattorini
11:15-12:00 Guest Lecture 12: Interferon Gamma Release Assays: advantages, limitations,
new advances
Delia Goletti (Italy)
12:00-12:15
Dormancy and resuscitation in Mycobacterium tuberculosis (OP 185)
Barbara Tizzano, Matthias Wilmanns, Stefan Niemann
12:15-12:30
Immunological features in children with tuberculosis (OP 234)
Anna Starshinova, Natalia Korneva, Julia Ovchinnikova, Olga Yakunova, Elena Potapenko,
Irina Dovgaluk
12:30-13:00 Poster Price Awards
13:00-13:15 Closing Remarks
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INVITATION 2014
Dear Colleagues!
The 35th Annual Congress of the European Society of Mycobacteriology (ESM) will take
place from 29 June - 2 July 2014 in the city of vienna, Austria. While in the aftermath of
World War I and the collapse of the Austrian-Hungarian-Empire, vienna was sadly acting
as the capital of tuberculosis in Europe, today this booming city of 1.7 Million population
is rightly proud of its high standards in „quality of Living“. The event will address the
many challenges facing key areas in mycobacteriology and will also especially focus on
Mycobacterium capraeandpublichealth.Thelecturesandseminarswillpromotescientific
exchange and also provide a forum for professional discussion with colleagues and with
the industry in a relaxed atmosphere. Planning is already underway and the website of the
European Society of Mycobacteriology will be regularly updated with new information. We
arelookingforwardtoyourscientificcontributionswithinterestandtowelcomingyouto
another exciting and informative ESM annual meeting.
Univ.-Prof. Dr. Franz allerberger
Chair
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LIST OF LECTURES
Lecture 1
TOWARDS GENOMIC EPIDEMIOLOGY OF MYCOBACTERIUM TUBERCULOSIS
Philip Supply
Lecture 2
DEALING WITH UNCERTAINTIES - INTERPRETING GENOMIC DATA FOR PUBLIC
HEALTH ACTION
Timothy Walker
Lecture 3
Advances in molecular typing of M. tuberculosis: should WGS become the
gold standard?
Dick van Soolingen
Lecture 4
Bench Top Next generation sequencing: Revolutionizing molecular
epidemiology and diagnostics?
Stefan Niemann
LIST OF GUEST LECTURES
Guest Lecture 1
NEW INSIGHTS INTO PATHOGENOMICS OF THE TUBERCULOSIS AGENT
Roland Brosch
Guest Lecture 2
Molecular epidemiology of tuberculosis spreading in European
metropolitan areas
Andrea Gori
Guest Lecture 3
Rapid whole genome sequencing as a tool to manage MDR/XDR-TB patients
Philip Butcher
Guest Lecture 4
LESSONS ON DRUG RESISTANCE FROM WGS OF ONE THOUSAND CLINICAL ISOLATES
Taane Clark
Guest Lecture 5
Low density microarrays as potential tools for the diagnosis of drug
resistant tuberculosis
Paolo Miotto
Guest Lecture 6
Molecular and quantitative approaches to DST – tools towards
better guided TB therapy in the days of MDR and XDR
Erik Christian Boettger
14
Guest Lecture 7
Challenges of pyrazinamide testing
Sven Hoffner
Guest Lecture 8
New approaches to tuberculosis treatment
Christian Lienhardt
Guest Lecture 9
Host-based biomarkers and different outcomes of MTB infection
Gerhard Walzl
Guest Lecture 10
New TB vaccines, where we are and where we go
Carlos Martin
Guest Lecture 11
New challenges for Buruli ulcer control
Françoise Portaels
Guest Lecture 12
Interferon Gamma Release Assays: advantages, limitations, new
advances
Delia Goletti
Honoree lecture
TUBERCULOSIS: NEW DRUGS ON THE HORIZON
Giovanna Riccardi
Gertrud Meissner Award Lecture
CAUSES AND CONSEQUENCES OF PARALLEL EVOLUTION OF TUBERCULOSIS AND
HUMANS OVER SEVENTY THOUSAND YEARS
Iñaki Comas
15
LIST OF ORAL PRESENTATIONS (OP)
M. TUBERCULOSIS GENOMICS I
OP 39
IDENTIFICATION OF LINEAGE-SPECIFIC GENETIC MARKERS FROM MORE THAN 1,500
MTBC ISOLATES
Francesc Coll, Kim Mallard, Mark D. Preston, Stephen Bentley, Julian Parkhill, Ruth McNerney,
Nigel Martin, Taane G Clark
OP 157
COMPARATIVE TRANSCRIPTOMIC ANALYSIS OF MyCOBaCTERIUM TUBERCULOSIS IN
A LIPID ENVIRONMENT
Patricia Del Portillo, Maria Jesus Garcia, Jorge Gonzalez-y-Merchand,
Maria Mercedes Zambrano, Juan Germán Rodríguez, Juan Manuel Anzola
OP 53
INSIGHTS INTO THE GENE ACTIVITY OF MyCOBaCTERIUM TUBERCULOSIS GROWING
IN A FATTY-ACID ENRICHED MEDIUM
Juan German Rodriguez, Adriana Carolina Hernandez, Jorge Gonzalez-y-Merchand,
Addy Cecilia Helguera-Repetto, Jose Ricardo Bustos, Juan Manuel Anzola,
Maria Mercedes Zambrano, Patricia del Portillo, Maria Jesus Garcia
OP 246
GENERATION AND CHARACTERIZATION OF A MyCOBaCTERIUM TUBERCULOSIS DELETION MUTANT DEFICIENT IN A PUTATIVE SUBUNIT OF A HETERODIMERIC ABC MULTIDRUG TRANSPORTER
Sven Malm, Silvia Maaß, Stefan Ehlers, Stefan Niemann
OP 164
CHARACTERIZATION OF THE SMALL RNA ExPRESSION PROFILES INDUCED BY ANTIBIOTICS IN MyCOBaCTERIUM TUBERCULOSIS
Paolo Miotto, Matthias Merker, Barbara Tizzano, Davide Cittaro, Dejan Lazarevic, Elia Stupka,
Stefan Niemann, Daniela Maria Cirillo
OP 23
UNDERSTANDING THE BIOLOGY OF POLY-RIFAMPICIN RESISTANCE IN MyCOBaCTERIUM TUBERCULOSIS
Margaretha de vos, Gail Erika Louw, Rob Warren, Thomas victor, Paul van Helden
16
OP 260
WHOLE GENOME SEqUENCING FROM EARLY CULTURE OR DIRECTLY FROM CLINICAL
SAMPLES
Mathilde Mairey, Bénédicte Condamine, Christine Hubans-Pierlot, Stéphanie Ferreira,
Philip Supply, Caroline Allix-Béguec
OP 245
GENOME WIDE ASSOCIATION ANALYSIS OF TUBERCULOSIS RESISTANCE IN DAIRY
CATTLE
Mairead Bermingham, Steve Bishop, John Woolliams, Adrian Allen, Stewart McBride,
David Wright, Jon Ryder, Robin Skuce, Stanley McDowell, Liz Glass
MOLECULAR EPIDEMIOLOGY OF TUBERCULOSIS
AND STRATEGY FOR CONTROL
OP 47
SUCCESSFUL RUSSIAN CLONE OF MyCOBaCTERIUM TUBERCULOSIS: ORIGIN, EMERGENCE AND CURRENT SPREAD
Igor Mokrousov
OP 73
LARGE SEqUENCE POLYMORPHISMS OF LATIN AMERICAN-MEDITERRANEAN (LAM)
MyCOBaCTERIUM TUBERCULOSIS STRAINS ISOLATED IN ITALY AND MOLECULAR
EPIDEMIOLOGY OF THE LAM RDRIO STRAINS
OP 224
TRACING MyCOBaCTERIUM TUBERCULOSIS TRANSMISSION: WHEN RESOLUTION
MATTERS
Thomas Kohl, Roland Diel, Stefan Niemann
OP 43
DETECTION OF RIFAMPICIN RESISTANCE IN MyCOBaCTERIUM TUBERCULOSIS BY
PADLOCK PROBES AND A MAGNETIC NANOBEAD-BASED READOUT
Anna Engström, Teresa Zardán Gómez De La Torre, Maria Strømme, Mats Nilsson,
David Herthnek
OP 89
SPRINT: SPOLIGO-RIFAMPICIN-ISONIAZID TYPING
Michel Gomgnimbou, Ivan Hernández-Neuta, Stefan Panaiotov, Elizabeta Bachiyska,
Palomino Juan Carlos, Anandi Martin, Patricia Del Portillo, Guislaine Refrégier, Christophe Sola
17
OP 154
DEVELOPMENT OF AN In vITRO SYSTEM TO PERFORM TIME-KILL CURVES OF LEVOFLOxACIN AGAINST MyCOBaCTERIUM TUBERCULOSIS
Aline Barth, Robert May, Judith Johnson, Charles Peloquin, Hartmut Derendorf
OP 212
RAPID TESTING FOR DRUG SUSCEPTIBILITY
Antonino Catanzaro, Timothy Rodwell, Camilla Rodrigues, valeriu Crudu, Thomas victor, Kanchan Ajbani, Elena Romancenco, Andre Trollip, Cindy Hayes, Roberta Lynn Jackson, Theodore
Ganiats, Erik Groessl, Richard Garfein
OP 229
KANAMYCIN, AMIKACIN, CAPREOMYCIN CROSS RESISTANCE IN MyCOBaCTERIUM
TUBERCULOSIS ISOLATES FROM ROMANIA
Daniela Homorodean, Jodal Andrea Melinda, Sven Hoffner
OP 243
EPIDEMIOLOGICAL TRENDS IN TB AND M/xDR-TB AND PROGRESS ON THE IMPLEMENTATION OF THE CONSOLIDATED ACTION PLAN TO PREVENT AND COMBAT M/xDR-TB
IN THE WHO EUROPEAN REGION
Kristin Kremer, Andrei Dadu, Martin van den Boom, Pierpaolo de Colombani, Masoud Dara
OP 244
EMERGENCE OF xDR IN HIGH BURDEN MDRTB COUNTRY: A THREAT TO PUBLIC
HEALTH AND TB CONTROL PROGRAM
Elena Romancenco, valeriu Crudu, Ecaterina Noroc, Nadejda Turcanu, Liliana Domente,
SofiaAlexandru
OP 96
MyCOBaCTERIUM TUBERCULOSIS BEIJING GENOTYPE STRAINS ARE CAPABLE OF
RESISTING TRANSIENT ExPOSURE TO RIFAMPICIN, AS STUDIED IN A NOVEL MICROCOLONY APPROACH
Alice den Hertog, Sandra Menting, Dick van Soolingen, Richard Anthony
NEW THERAPEUTIC REGIMENS FOR TB AND MDR-TB
OP 46
ANTI-TUBERCULOSIS ACTIVITY OF EFFLUx INHIBITORS AGAINST DRUG RESISTANT
MyCOBaCTERIUM TUBERCULOSIS
Diana Machado, Isabel Couto, Jorge Ramos, Miguel viveiros
OP 66
ORAL PHENYLBUTYRATE AND VITAMIN D3 INDUCE THE CATHELICIDIN LL-37 IN IMMUNE CELLS: A DOSE FINDING STUDY FOR TREATMENT OF TUBERCULOSIS
Rubhana Raqib, Akhirunnesa Mily, Rokeya Rekha, S.M. Mostafa Kamal, Protim Sarker,
Zeaur Rahim, Gudmundur Gudmundsson, Birgitta Agerberth
18
OP 225
STERILIZING ACTIVITY OF DRUG COMBINATIONS AGAINST ACTIVELY REPLICATING
AND NONREPLICATING MyCOBaCTERIUM TUBERCULOSIS
Giovanni Piccaro, Federico Giannoni, Donatella Pietraforte, Alessandro Mustazzolu,
Lanfranco Fattorini
NONTUBERCULOUS MYCOBACTERIA
OP 64
WHAT IS THE ROLE OF HORIZONTAL GENE TRANSFER IN MYCOBACTERIAL DRUG RESISTANCE AND VIRULENCE?
Sylvia Cardoso Leão, Katiane Santin, Christiane Lourenco Nogueira, Paulo Jose Martins Bispo,
Cristianne Kayoko Matsumoto
OP 75
MOLECULAR CHARACTERIZATION OF MyCOBaCTERIUM avIUM CLINICAL STRAINS
FROM ST. PETERSBURG, RUSSIA
Daria Starkova, Tatiana Otten, Igor Mokrousov, Anna vyazovaya, Boris vishnevskiy,
Olga Narvskaya
OP 113
WHOLE GENOME SEqUENCING PROVIDES EVIDENCE FOR TRANSMISSION OF MyCOBaCTERIUM aBSCESSUS BETWEEN CYSTIC FIBROSIS PATIENTS
Josephine M. Bryant, Dorothy M. Grogono, Daniel Greaves, Juliet Foweraker, Iain Roddick,
Thomas Inns, Mark Reacher, Charles S. Haworth, Martin D. Curran, Simon R. Harris,
Sharon J. Peacock, Julian Parkhill, R. Andres Floto
OP 159
EMERGENCE OF CLINICALLY RELEVANT NON-TUBERCULOUS MYCOBACTERIAL INFECTIONS IN SAUDI ARABIA
Bright varghese, Ziad Memish, Naela Abouljadayel, Raafat Al-Hakeem, Fahad Alrabiah,
Sahal Al-Hajoj Al-Nakhli
IGRA AND LATENT TB
OP 185
DORMANCY AND RESUSCITATION IN MyCOBaCTERIUM TUBERCULOSIS
Barbara Tizzano, Matthias Wilmanns, Stefan Niemann
OP 234
IMMUNOLOGICAL FEATURES IN CHILDREN WITH TUBERCULOSIS
Anna Starshinova, Natalia Korneva, Julia Ovchinnikova, Olga yakunova, Elena Potapenko,
Irina Dovgaluk
19
LIST OF POSTER PRESENTATIONS (P)
M. TUBERCULOSIS GENOMICS
P 24
FITNESS OF MyCOBaCTERIUM TUBERCULOSIS WITH MUTATIONS IN THE RPSL, RRS,
GIDB AND RPOB GENES
Fernanda Spies, Andrea von Groll, Andrezza Ribeiro, Daniela Ramos, Marta Ribeiro, Anandi
Martin, Juan Carlos Palomino, Maria Lucia Rossetti, Pedro Almeida da Silva, Arnaldo Zaha
P 42
DECIPHERING THE EVOLUTIONARY HISTORY OF THE M. TUBERCULOSIS LATIN
AMERICAN-MEDITERRANEAN (LAM) GENOTYPE
Anzaan Dippenaar, Rob Warren, Nico Gey van Pittius
P 44
CHARACTERIZATION OF ExTENSIVELY DRUG-RESISTANT MyCOBaCTERIUM
TUBERCULOSIS IN SIBERIA
Maya Dymova, Olga Alkhovik, Andrey Cherednichenko, Tatyana Petrenko, Maxim Filipenko
P 90
LABEL-FREE AND MULTIPLExED ELECTROCHEMICAL DETECTION OF PCR
FRAGMENTS ON SCREEN-PRINTED ELECTRODE ARRAYS. APPLICATION TO
SPOLIGOTYPING OF MyCOBaCTERIUM TUBERCULOSIS
viet Hai Le, Steeve Reisberg, Michel Gomgnimbou, Christophe Sola, Minh Chau PHAM, Benoit
Piro
P 94
MOLECULAR IDENTIFICATION OF MyCOBaCTERIUM TUBERCULOSIS COMPLEx
SUBSPECIES IN PROVINCE OF MODENA, ITALY
Giulia Fregni Serpini, Sara Tagliazucchi, Giulia Forbicini, Nadia Nanni, Rita Magnani, Anna
Fabio, William Gennari, Anna Maria Teresa Sabbatini, Antonella Grottola, Monica Pecorari
P 108
UNUSUAL LARGE-SCALE CHROMOSOMAL REARRANGEMENTS IN MyCOBaCTERIUM
TUBERCULOSIS BEIJING B0/W148 CLUSTER ISOLATES
Egor Shitikov, J Bespyatykh, D Ischenko, I Karpova, E Kostryukova, y Isaeva, E Nosova, A
vyazovaya, I Mokrousov, O Narvskaya, B vishnevsky, T Otten, v Zhuravlev, P yablonsky, E
Ilina, v Govorun
P 123
DETECTION OF MUTATIONS BEYOND THE ‘HOT-SPOT’ REGIONS OF FIRST- AND
SECOND-LINE DRUG RESISTANCE GENES IN ExTENSIVELY DRUG-RESISTANT
MyCOBaCTERIUM TUBERCULOSIS ISOLATES USING WHOLE GENOME SEqUENCING
Asho Ali, Zahra Hasan, Taane Clark, Ruth McNerney, Kim Mallard, Mridul Nair, Arnab Pain,
Rumina Hasan
20
P 248
COMPARISON OF TWO COMMERCIAL MOLECULAR ASSAYS FOR THE DETECTION OF
TUBERCLE BACILLI IN PARAFFIN-EMBEDDED TISSUES
Nataša Fajfar, Manca Zolnir Dovc
P5
PREVALENCE OF HAARLEM FAMILY IN MyCOBaCTERIUM TUBERCULOSIS IN WORLD
POPULATION: SYSTEMATIC REVIEW AND META-ANALYSIS
Rashid Ramazanzadeh, Daem Roshani
P 26
GENOTYPING OF MyCOBaCTERIUM BOvIS FROM FARMED ELK IN KOREA BY
SPOLIGOTYPING AND VARIABLE NUMBER TANDEM REPEAT ANALYSIS
Jae Myung Kim, yun-Ho Jang, yoonra Jang, Soyoon Ryoo, Narae Kim, Jae Min Jang, ShinSeok Kang, Hyun Sub Byun, Suk Chan Jung
P 48
MOLECULAR CHARACTERISTICS OF MyCOBaCTERIUM TUBERCULOSIS BEIJING
GENOTYPE ISOLATES FROM RUSSIA
Anna vyazovaya, Igor Mokrousov, Natalia Solovieva, Tatiana Otten, Boris vishnevskiy, Olga
Narvskaya
P 61
MyCOBaCTERIUM TUBERCULOSIS INFECTION IN CATTLE IN CROATIA - CASE
REPORTS
Silvio Spicic, vera Katalinic-Jankovic, Ivana Racic, Maja Zdelar-Tuk, Sanja Duvnjak, Zeljko
Cvetnic
P 67
TRANSMISSION OF M. BOvIS INFECTION AMONG WILD ANIMALS
Marek Lipiec, Monika Krajewska
P 71
THE USEFULNESS OF ELISA TEST FOR THE DIAGNOSIS OF BOVINE TUBERCULOSIS IN
POLISH CONDITIONS
P 76
MIRU-VNTR TYPING REVEALS HIGH HETEROGENEITY IN A SUPPOSEDLY CLONAL
GROUP OF MyCOBaCTERIUM BOvIS
Sabrina Rodriguez-Campos, yurena Navarro, Beatriz Romero, Javier Bezos, Lucía de Juan,
Carmen Casal Comendador, Lucas Domínguez, Alexandra Gutiérrez, Darío García de viedma,
Alicia Aranaz
21
P 77
Evaluation of COBAS® TaqMan MTB for direct detection of
Mycobacterium tuberculosis complex in comparison with the COBAS®
Amplicor MTB
Claudia Ritter, Guido Bloemberg, Antje Voit, Vanessa Deggim, Erik Böttger
P 79
A putative compensatory mutation in the rpoC-gene exclusively detected
in isolates of the Resistant European Tuberculosis (RET) cluster with a
MDR-TB phenotype
Jessica De Beer, Indra Bergval, Anja Schuitema, Richard Anthony, Maryse Fauville, Beatriz
Ferro, Viviana Ritacco, Aldert Zomer, Jakko van Ingen, Dick van Soolingen
P 87
Current status of bovine tuberculosis in Poland - 3 years after the
recognition of the country free of the disease
P 106
TRAINING IN BIOSAFETY AGAINST M. TUBERCULOSIS: EXPERIENCE IN THE
MYCOBACTERIA REFERENCE CENTER AT THE UNIVERSITY OF CORDOBA, SPAIN
Vaquero-Alvarez Esther, Pablo López-Roldan, Emilio J Aguilar, Fernando Palomares, Francisco
Torralbo, Manuel Vaquero, Manuel J Casal
P 110
A novel model to control for patient risk factors in measuring the
transmissibility of Mycobacterium tuberculosis strains and lineages
Hanna Nebenzahl-Guimaraes, Martien Borgdorff, Jessica de Beer, Megan Murray, Dick van
Soolingen
P 125
Epidemiological analyses of tuberculosis in Archangelsk, Russia and
implementation of a rapid assay for detection of resistance in this high
burden setting
Platon Eliseev, Andrey Maryandyshev, Elena Nikishova, Irina Tarasova, Galina Gorina, Erja
Chryssanthou, Lars-Olof Larsson, Malin Ridell
P 139
Accurate and efficient identification of drug resistance and local MDR
cluster strains of Mycobacterium tuberculosis using an adapted SNPbased multiplex LPA assay
Sarah Sengstake, Indra Bergval, Anja Schuitema, Kiki Tuin, Edgar Abadia, Jessica De
Beer, Nino Bablishvili, Nino Bzekalava, Nadia Brankova, Viktoria Levterova, Rusudan
Aspindzelashvili, Christophe Sola, Stefan Panaiotov, Dick van Soolingen, Richard Anthony
P 149
Genotypic diversity of Mycobacterium tuberculosis in conjunction
with demographic and epidemiologic data underlines major differences
between French versus Foreign-born cases in the Rhône-Alpes region,
France (2000-2010)
Catherine Pichat , David Couvin, Gérard Carret, Véronique Jacomo, Anne Carricajo, Sandrine
Boisset, Jean-Pierre Flandrois, Gérard Lina, Nalin Rastogi
22
P 152
Molecular analysis of Mycobacterium bovis isolates from humans in
Italy: comparison with the genotype database of animal strains
Maria Pacciarini, Maria Goria, Giuseppina Ferraro, Silvia Tagliabue, Ester Mazzola, Maria Tullia
Simonetti, Paola Dal Monte, Piera Mazzone, Maria Beatrice Boniotti
P 153
Changes in Mycobacterium tuberculosis population structure in the
French Departments of the Americas: a 17 years overview
Julie Millet, Elisabeth Streit, Anne Gaël Bomer, Franzisca Schuster, Nalin Rastogi
P 171
Populational-based survey of molecular and epidemiological links
between human and animal infections by Mycobacterium bovis
Beatriz Romero, Juan José Palacios, Yurena Navarro, María Francisca Copano, Lucía de Juan,
Julio Alvarez, Tatiana Alende, Lucas Domínguez, Darío García de Viedma
P 175
Retrospective analysis for diagnosis of Mycobacterium tuberculosis
Huseyin Guducuoglu, Abdullah Bektas
P 176
European Union Reference Laboratory for Bovine Tuberculosis: an
useful tool towards harmonization of protocols
Beatriz Romero, Javier Bezos, Carmen Casal Comendador, Julio Alvarez, Francisco Lozano,
Nuria Moya, Lucía de Juan
P 197
Rapid diagnosis, molecular typing patterns and drug susceptibility
profiles of mycobacteria isolated from patients with tuberculous
meningitis in Ankara
GÜLNUR TARHAN, Hülya Şimşek, Salih CESUR, Ismail CEYHAN
P 198
Population structure of Mycobacterium tuberculosis in two
geographically distant areas of Argentina
Johana Monteserin, Ana Etchart, Ana Reniero, Beatriz López, Viviana Ritacco
P 214
Performance of Spoligotyping in extrapulmonary tuberculosis
diagnosis in patients attending a tertiary care hospital
Wellman Ribon, Magda Lorena Orduz Zambrano
P 219
GENETIC DIVERSITY OF MYCOBACTERIUM TUBERCULOSIS COMPLEX STRAINS
ISOLATED IN THE NORTH-WESTERN AND CENTRAL COUNTIES OF ROMANIA
IONELA SORINA MUNTEAN, Adriana Drăgan, Daniela Homorodean, ANDREEA JODAL, Sven
Hoffner
23
P 220
Molecular genotyping confirms tuberculosis infection transmission
in household contacts as the main infection transmission route among
children
Viesturs Baumanis, Iveta Ozere, Inta Jansone, Anda Nodieva, Ilva Pole, Matiss Bauskenieks,
Girts Skenders
P 227
Whole Genome Sequencing Reveals Local Transmission Patterns of
Mycobacterium bovis in Sympatric Cattle and Badger Populations
Roman Biek, Anthony O‘Hare, David Wright, Tom Mallon, Carl McCormick, Richard Orton,
Stanley McDowell, Hannah Trewby, Robin Skuce, Rowland Kao
P 230
Multidrug resistant tuberculosis epidemic in Swaziland: ongoing
transmission of multidrug resistant Mycobacterium tuberculosis
strains
M. Merker, E. Sanchez, P. Beckert, F. Jochims, M. Bonnet, Th. Dlamini, M. Bastard, H.
Koarakozian, S. Rüsch-Gerdes, S. Niemann
P 231
TB-miner, an on-line tool to describe classification of M. tuberculosis
complex based on the wide scale diversity of the Netherlands database
Jérôme Azé, Guislaine Refrégier, Kristin Kremer, Dick van Soolingen, Christophe Sola
P 241
Distribution and Spatial Analysis of Foci of Tuberculosis in Cattle and
Deers in the South of Beira Interior (Portugal)
Luis Caiola Ribeiro, Paulo Fernandez, Manuel Martins
P 242
Tuberculosis in the Czech Republic at risk groups: Casuistry from the
current time
Maria Müllerová, M. VašákováE. Kopecká, J. Homolka, J. Pohl, K. Křepela
P 250
Correlation between streptomycin intermediate-level resistance and
gidB mutation in an endemic multidrug-resistant tuberculosis cluster
Joao Perdigao, Rita Macedo, Diana Machado, Carla Silva, Luisa Jordao, Isabel Couto, Miguel
Viveiros, Isabel Portugal
P 257
Danish Mycobacterium tuberculosis outbreak strain is spreading
among Greenlandic Inuit in Denmark and Greenland
Troels Lillebaek, Ase Bengard Andersen, Erik M. Rasmussen, Zaza Kamper-Jørgensen,
Matthias K. Pedersen, Karen Bjoern-Mortensen, Karin Ladefoged, Vibeke Ø. Thomsen
P 258
Active transmission of Mycobacterium tuberculosis (Mt) continues at
surprisingly high rates in Denmark
Troels Lillebaek, Åse Bengård Andersen, Niels Jørgen Seersholm, Vibeke Østergaard Thomsen
24
MDR DIAGNOSTIC AND DST
P 15
RESISTANCE PATTERNS TO SECOND-LINE DRUGS IN MyCOBaCTERIUM
TUBERCULOSIS IN SPAIN
Pilar Ruiz, Juan B Gutierrez, Manuel Causse, Manuel J Casal
P 27
EVALUATION THE BACT ALERT 3D SYSTEM FOR RECOVERY AND IDENTIFICATION OF
MYCOBACTERIA FROM CLINICAL SAMPLES
María Rosarys Martínez Romero, Misleidis Sardiña, Grechen García, Lilian María Mederos
P 28
DETECTION OF AMIKACIN RESISTANCE IN MyCOBaCTERIUM TUBERCULOSIS USING
THE NITRATE REDUCTASE ASSAY AND THE RESAZURIN MICROTITRE ASSAY
Dihadenys Lemus, Carlos Washington Reyes, Miguel Echemendia, Juan Carlos Palomino,
Anandi Martin
P 37
GENOMIC MUTATION PROFILING OF MULTIDRUG RESISTANCE TUBERCULOSIS
ISOLATES USING BY NOVEL GENOTYPE® MTBDRPLUS ASSAY
Anand Kumar Maurya, Surya Kant, Amresh Kumar Singh, Ram Awadh Singh Kushwaha, Manoj
Kumar, vijaya Lakshmi Nag, Tapan N Dhole
P 69
FIELD EVALUATION OF THE DIRECT COLORIMETRIC NITRATE REDUCTASE ASSAY FOR
THE SIMULTANEOUS DETECTION OF MDR- AND xDR-TB IN ARGENTINA
Belen Imperiale, Nora Morcillo, Juan Carlos Palomino, Anandi Martin
P 80
EVALUATION OF THE AID TB RESISTANCE LINE PROBE ASSAY FOR RAPID DETECTION
OF GENETIC ALTERATIONS ASSOCIATED WITH MyCOBaCTERIUM TUBERCULOSIS
DRUG RESISTANCE
Claudia Ritter, Katja Lucke, Frick Sirgel, Robin Warren, Erik Böttger, Guido Bloemberg
P 85
A NEW COLORIMETRIC PLATE FOR THE RAPID DIAGNOSIS OF ExTENSIVELY DRUGRESISTANT TUBERCULOSIS DIRECTLY IN SPUTUM SAMPLES
Beatriz Lopez, Lucia Barrera, Martha Ambroggi, Susana Poggi, Peter vandamme, Juan Carlos
Palomino, viviana Ritacco, Anandi Martin
P 95
RPOB POLYMORPHISMS IN MyCOBaCTERIUM TUBERCULOSIS COMPLEx FROM A
POPULATION IN GUINEA-BISSAU
AF Sutre, A Sanca, A Mané, v Henriques, C Portugal, Luisa Sancho, A Cardoso, E Paixão, CqF
Leite, JI Salem, A Antunes, EL Duarte, S David
25
P 121
EXPAND-TB: Experience of establishing TB laboratories in limited
resources settings in Eastern Europe and Central Asia
Alexei Korobitsyn; K. Kao, C.N. Paramasivan, D. Orozco
P 131
Comparative Evaluation of TK SLC-L, the Rapid Liquid Mycobacterial
Culture Medium, with BACTEC MGIT
İhsan Hakkı Çiftçi, Engin Karakeçe
P 138
Development of a new DNA microarray platform for the detection of
MDR tuberculosis
Andrea M. Cabibbe, Klaus Reither, Daniela M. Cirillo
P 140
Evaluation of phenotypic and genotypic drug susceptibility testing
methods for fluoroquinolone resistance in M. tuberculosis
Nele Coeck, Bouke de Jong, Leen Rigouts
P 141
TB PAN NET consortium: shared databases for the characterization of
mutations involved in drug-resistant M. tuberculosis phenotype
Andrea M. Cabibbe, Paolo Miotto, Emanuele Borroni, Ilaria C. Valente, Silke Feuerriegel,
Petras Stakenas, Ewa Augustynowicz-Kopeć, Vanessa Mathys, Sven Hoffner, Stefan Niemann,
Daniela M. Cirillo
P 142
Dynamic microcolony growth monitoring for drug susceptibility
testing of ethambutol and pyrazinamide
Alice den Hertog, Sandra Menting, Ernst Smienk, Sarah Sengstake, Sven Hoffner, Richard
Anthony
P 144
pncA gene mutations and pyrazinamide resistance in Swedish multidrugresistant tuberculosis 2003-2013
Mikael Mansjö, Jim Werngren, Sven Hoffner, Ylva Lidén
P 160
The Influence of Glutoxim on the Resistance of Mycobacterium
tuberculosis (Mtb) Strains to Isoniazid
Olga Manicheva, Natalya Solovyeva, Viacheslav Zhuravlev, Victor Antonov, Dmitriy Aizikov,
Marina Shulgina
P 173
Genetic characterization of pyrazinamide-resistant M. tuberculosis
complex isolates in an Italian north-eastern area during a four year
period
Marta Peracchi, Loredana Fallico, Marta Viero, Mario Rassu, Michela Pascarella, Riccardo
Manganelli, Giorgio Palù
26
P 174
INTEGRATING THE XPERT MTB/RIF ASSAY IN A DIAGNOSTIC WORK FLOW FOR RAPID
DETECTION OF MYCOBACTERIUM TUBERCULOSIS IN A LOW PREVALENCE AREA
Akos Somoskovi, Claudia Ritter, Vanessa Deggim, Antje Voit, Eric C. Böttger, Guido V.
Bloemberg
P 179
The Efficiency of a New Decontamination and Concentration Kit which
Eliminates Centrifugation, in Isolation of Mycobacteria from Sputum
Samples
P 180
Rapid diagnosis of multi- and extensively drug resistant tuberculosis
among patients with high risk of TB resistance
Valeriu Crudu, Elena Romancenco, Ecaterina Noroc, Nadejda Turcan, Galina Blagoadeteleva
P 181
Evaluation of second-line antituberculosis drugs susceptibility testing
of multidrug-resistant (MDR) isolates of Mycobacterium tuberculosis
using Etest
Hulya Simsek, Gülnur Tarhan, Salih Cesur
P 182
Evaluation of the presence of mycobacteria belonging to the
Mycobacterium tuberculosis complex in animal tissues by real-time PCR
Pedro Costa, Ana Ferreira, Ana Amaro, Isabel Couto, Miguel Viveiros, João Inácio
P 193
Evaluation of the Xpert MTB/RIF assay for the diagnosis of tuberculosis
Ali Albay, Ozgul Kisa, Mustafa Guney, Gokselin Dogan, Kemal Tekin
P 199
Rapid detection of M.tuberculosis drug resistance to second line
antibiotics in sputum samples by using the multi-competitive allelespecific real-time PCR
Yulia Alyapkina, Michail Vladimyrsky, Marina Lapenkova, Dmitry Varlamov
P 204
Diagnosis of Pulmonary Tuberculosis using GenoType MTBDR Assay in HIVinfected Patients, Tg.-Mures, Romania
Lilla Lőrinczi, Maria Nemes, Zaharia Kézdi Erzsébet Iringó, Mihaela Patraulea, Székely Edit,
Vas Krisztina Eszter, Lőrinczi Zoltán
P 232
The possibility of early correction of chemotherapy for pulmonary
tuberculosis on the basis of molecular genetic methods
Mariya Pavlova, Viatcheslav Zhuravlev, Ludmila Archakova, Nadezhda Sapozhnikova, Natalya
Solovyeva, Anna Starshinova
27
P 253
THE CORRELATION BETWEEN PHENOTYPIC AND GENETIC RESISTANCE TO
ETHAMBUTOL IN PATIENTS FROM MOSCOW REGION
Ruslan Ludannyy, Maria Alvarez Figueroa, Anastasiya Prokopenko
P 256
MDR TB WITH MIxED STRAINS: A CHALLENGE IN DIAGNOSTIC AND TREATMENT OF
PATIENTS
valeriu Crudu,ElenaRomancenco,EcaterinaNoroc,NadejdaTurcan,LilianaDomente,Sofia
Alexandru, Dumitru Chesov, Stefan Niemann, Matthias Merker, Sabine Rüsch-Gerdes, Antonino
Catanzaro
P 259
ExTERNAL qUALITY CONTROL FOR DRUG SUSCEPTIBILITY TESTING IN ROMANIAN
TUBERCULOSIS LABORATORY NETWORK
Daniela Homorodean, Andreea Melinda Jodal
P 262
LATE-PCR DETECTION OF M(x)DR-TB USING FLUORESCENT SIGNATURES
L. Rice, J.Rice, L. Wangh; N. Kurpina, B. Kreiswirth, N. Casali, F. Drobniewski; M. De vos, R.
Warren
P 161
EFFICIENCY OF CHEMOTHERAPY OF xDR TB PATIENTS WITH LINEZOLIDE
Anastasia Samoilova, Irina vasilyeva, Tatev Bagdasarian, Marina Burakova, Svetlana
Moiseyeva
TB BIOMARKERS AND NOVEL VACCINE STRATEGIES
P 21
DRUG TOLERANCE TO ETHAMBUTOL OF MyCOBaCTERIUM TUBERCULOSIS CELL
WALL DEFICIENT L-FORMS
Georgi Slavchev, Nadya Markova
P 86
MALARIA, TUBERCULOSIS, HIV and HBV COINFECTIONS IN A RURAL POPULATION IN
THE SOUTH OF GHANA
Natalia Julca, Alejandra Martinez-Serna, Maria Carmen Menendez, Carolina Garrido, Patricia
Marin-Garcia, Antonio Puyet, Jose Manuel Bautista, Carmen De Mendoza, Maria Jesus Garcia,
Amalia Diez
P 205
CIRCULATING MIRNA SIGNATURES IN TUBERCULOSIS
Paolo Miotto, Grace Mwangoka, Ilaria C. valente, Luca Norbis, Giovanni Sotgiu, Alessandro
Ambrosi, Luigi Codecasa, Delia Goletti, Alberto Matteelli, Klaus Reither, Daniela M. Cirillo
28
P 207
IDENTIFICATION OF ROUTINE CLINICAL MYCOBACTERIA ISOLATES WITH MALDI-TOF
MS USING THE NEW BRUKER MYCOBACTERIA LIBRARY
Gorkem yaman, Cigdem Celikkan, Derya Turk
P 222
EVALUATION OF FLUOROTYPE MTB FOR DIRECT DETECTION OF MyCOBaCTERIUM
TUBERCULOSIS COMPLEx AND GENOTYPE MTBDRPLUS FOR DETERMINING OF
RIFAMPICIN AND ISONIAZID RESISTANCE
Gonca Erkose Genc, Dilek Satana, Esra yildrim, Zayre Erturan, yildiz yegenoglu, Meltem uzun
P 18
IN MExICAN MESTIZOS THE HLA-DRB1*01 ALLELE CONFERS GENETIC
SUSCEPTIBILITY TO LEPROMATOUS LEPROSY (LL)
Monica Escamilla-Tilch, Iris Estrada-García, Nora M Torres-Carrillo, Rosalío Ramos-Payán, M.
Isabel Salazar, Mary Fafutis, Roberto Arenas-Guzmán, Sergio Estrada Parra, Julio Granados
P 41
MYCOBACTERIA IDENTIFICATION FROM PATIENTS WITH RESPIRATORY SYMPTOMS
ADMITTED IN PEDRO KOURÍ INSTITUTE, HAVANA, CUBA
María Rosarys Martínez Romero, Misleidis Sardiña, Grechen García, Lilian María Mederos,
yoandra Abad
P 51
A CASE OF MyCOBaCTERIUM TERRaE INFECTION IN CATTLE IN BOSNIA AND
HERZEGOVINA
Hajrudin Besirovic, Amer Alic, Silvio Spicic, Zeljko Cvetnic, Senad Prasovic
P 58
COMPARATIVE In vITRO ACTIVITIES OF RIFAMPIN, RIFAPENTINE AND RIFABUTIN
AGAINST MyCOBaCTERIUM KanSaSII
Michael Cynamon, Mary Sklaneyvamc
P 82
MyCOBaCTERIUM BRUMaE TRIGGERS CELL-CYCLE ARREST IN BLADDER CANCER
CELLS
Silvia Secanella-Fandos, Gemma Orpella-Aceret, Eduard Torrents, Alejandro Sánchez-Chardi,
Julia Lorenzo, Manuela Costa, Marina Luquin, Esther Julián
P 83
IN-DEPTH SURVEY OF MyCOBaCTERIUM avIUM SUBSP. paRaTUBERCULOSIS (MAP)
GENOTYPES IN GERMANY
Petra Möbius, Isabel Fritsch, Heike Köhler
29
P 84
Stability of genotyping targets in Mycobacterium avium subsp.
paratuberculosis (MAP) upon cultivation on different media, in vitro and
in vivo passage, and natural infection
Nadine Kasnitz, Heike Köhler, Petra Möbius
P 122
Prevalence of Mycobacterium avium subspecies paratuberculosis and
Mycobacterium avium subspecies hominissusis in biogas plants in the
Czech Republic
Monika Moravkova, Radka Pribylova, Iva Slana
P 129
Molecular analysis of human isolates belonging to Mycobacterium
avium complex collected during the years 2005-2010 in the Czech
Republic
Michal Slany, Vit Ulman, Eva Kalakayova, Iva Slana
P 130
Soil and plant contamination with M. a. paratuberculosis from tissues
of infected animals
Marija Kaevska, Samuel Lvoncik, Jiri Lamka, Iva Slana, Ivo Pavlik
P 134
Monitoring of Mycobacterium avium subsp. paratuberculosis survival
in fermented milk products by means of culture and quantitative real
time PCR methods
Barbora Klanicova, Iva Slana, Petr Roubal, Petr Kralik
P 145
Molecular analysis of Mycobacterium chelonae-M. abscessus group
isolates without conclusive species identification
Christiane Lourenco Nogueira, Cristianne Kayoko Matsumoto, Luciano Antonio Digiampietri,
João Carlos Setubal, Sylvia Cardoso Leão
P 150
Critical role of Toll-like Receptor 2 in Mycobacterium brumae-induced
antitumor activity
Silvia Secanella-Fandos, Carmen Fernández, Esther Julián
P 165
MYCOBACTERIAL INFECTIONS IN WILDLIFE: FIRST REVIEW FOR SLOVENIA
Mateja Pate, Urska Zajc, Darja Kusar, Diana Zele, Gorazd Vengust, Tina Pirs, Matjaz Ocepek
P 166
GenoType assay coupled with 16S rRNA and rpoB sequencing: a good
approach towards improved identification of nontuberculous
mycobacteria in a veterinary laboratory?
Mateja Pate, Darja Kusar, Urska Zajc, James Higgins, Patrick Camp, David Farrell, Doris Bravo,
Tod Stuber, Matjaz Ocepek
30
P 167
Non-pathogenic mycobacterium for the treatment of non-invasive
bladder cancer
Silvia Secanella-Fandos, Estela Noguera-Ortega, Hasier Eraña, Jofre Gasión, Marina Luquin,
Esther Julián
P 168
Specific formulation of mycobacteria improves its antitumor activity
against bladder cancer cells
Estela Noguera-Ortega, Núria Blanco-Cabra, Mónica Roldán, Marina Luquin, Esther Julián
P 191
PULMONARY TUBERCULOSIS CAUSED BY M. szulgai: CASE REPORT
Kemal Tekin, Ferhat Onur Ural, Mustafa Guney, Seyfettın Gumus, Ali Albay
P 195
Bloodstream Infection by Mycobacterium abscessus: A Case Report
Serhat Duyan, Mustafa Guney, Erman Ates, Abdullah Kilic, Ali Albay
P 203
PULMONARY DISEASE by NONTUBERCULOUS MYCOBACTERIA . A 10 YEARS STUDY
Maria Teresa Tortola Fernandez , Carmen Aleman, Meritxell Espuga, Israel Molina, Jose
Angel Rodrigo, Nuria Saborit, Asuncion Seminario, Teresa Soriano, Alba Torrent, Nuria MartinCasabona
P 213
Case Report of Mycobacterium tilburgii Infection
Timur S. Akpinar, O.K. Bakkaloglu, Burak Ince, O. Kaya Koksalan, Nesimi Buyukbabani, Bulent
Saka, Nilgun Erten, Zeki Kilicaslan, Cemil Tascioglu
P 216
The disability in leprosy due to social stigma
Hernando Yesid Estupiñán Velásquez, Debora Villa Villa, Wellman Ribon
P 218
PULMONARY MYCOBACTERIOSIS CAUSED BY MYCOBACTERIUM PEREGRINUM IN AN
OLD WOMAN
Ionela Muntean, Adriana Drăgan,Daniela Homorodean, Sven Hoffner
P 226
Molecular identification of Mycobacterium avium complex (MAC)
members recovered from clinical samples in a hospital in a 3-year period
Julio Alvarez, Beatriz Romero , Claudio Cuartero, Javier Bezos, Carmen Casal Comendador, N.
SánchezJohanna Gimeno, Lucas Domínguez, Enrique Gómez-Mampaso
P 228
Characterization of Mycobacterium in wild bird groups distributed in
the department of Santander, Colombia
Jhoner Rueda, Fernando Rondón, Wellman Ribon
31
P 235
MyCOBaCTERIUM aFRICanUM IN PORTUGAL: A CASE REPORT
Luisa Sancho, Clara Portugal, Luisa Tancredo, Maria Silva, Angela Dias, Filomena Silva,
Germano Sousa
P 239
OCCURRENCE OF MyCOBaCTERIUM avIUM SUBSP. paRaTUBERCULOSIS IN ROAD
KILLED WILD CARNIVORES IN PORTUGAL
Ana Matos, Luis Figueira, Maria Helena Martins, Manuel Martins, Filipa Loureiro, Maria de
Lurdes Pinto, Manuela Matos, Ana Cláudia Coelho
P 240
SEROSURVEY OF MyCOBaCTERIUM avIUM COMPLEx IN WILD BOARS IN PORTUGAL
Ana Matos, Luis Figueira, Maria Helena Martins, Manuel Martins, Manuela Matos, Maria de
Lurdes Pinto, Ana Claudia Coelho
P 254
TB OR MYCOBACTERIOSIS: DETECTION, SPECIES IDENTIFICATION AND DST IN
PRACTICE OF MICROBIOLOGY LAB
Tatiana Smirnova,ElenaLarionova,SofiaAndreevskaya,AlenaVorobyeva,LarisaChernousova
IGRA AND LATENT TB
P 118
INDETERMINATE qUANTIFERON-TB GOLD IN-TUBE RESULTS IN CHILDREN:
ASSOCIATION WITH PNEUMONIA?
Giulia Lombardi, Paola Dal Monte, Agnese Denicolò, Antonella Pace, Roberta Petrucci, Ilaria
Corsini, Maria Letizia Bacchi Reggiani, Salvatore Cazzato, Maria Paola Landini
P 233
CLINICAL AND ROENTGENOLOGICAL FEATURES IN CHILDREN WITH DIFFERENT
PROFILES OF IMMUNOLOGICAL TESTS
Anna Starshinova, Pavel Gavrilov, Natalia Korneva, Irina Dovgaluk
32
ABSTRACTS OF LECTURES (L)
L-1
TOWARDS GENOMIC EPIDEMIOLOGY OF
MyCOBaCTERIUM TUBERCULOSIS
Mathilde Mairey3, Caroline Allix-Béguec3, Philip
Supply1, 2, 3
1
Center for Infection and Immunity, InSERM 1019
CnRS UMR 8204, Lille, France
2
Institut pasteur de Lille, Lille, France
3
genoscreen, Lille, France
As a disruptive technology, next-generation sequencing
(NGS) opens the way towards comprehensive,
whole genome sequence (WGS)-sequence based
exploitation of microbial genetic information, for
improved diagnostics and disease surveillance.
However, several key issues need to be addressed for
relevant generalized use, in progressive replacement
of tuberculosis (TB) molecular diagnostics and
classical typing of Mycobacterium tuberculosis
isolates.
As an introduction to the workshop some of these
questionswillbebrieflyoverviewed,basedonrecent
findingsandourworkdoneintheframeoftheFP7funded Patho-NGen-Trace project, comprising
whole genome sequencing of > 1,000 isolates
of M. tuberculosis, Staphylococcus aureus and
Campylobacter jejuni. These questions include e.g.
the performances and scalability of NGS platforms,
the range of genome-wide variation to consider for
cluster detection using molecular tracing, the potential
and interest to detect other variations than single
nucleotide polymorphisms, the need to adapt NGS
strategies to the questions addressed (e.g. molecular
tracing vs detection of drug resistance), and the
need for new integrated, easy-to-use bio-informatics
pipelines and for a standardized genome-based
nomenclature system. The interest of WGS-based
approaches, to discover new, unexpected biological
features will also be illustrated on the basis of our
recent work done on particular human TB causing
strains,classifiedasM. canettii.
L-2
DEALING WITH UNCERTAINTIES - INTERPRETING
GENOMIC DATA FOR PUBLIC HEALTH ACTION
Timothy M Walker
University of Oxford, Oxford, UK
A growing body of literature suggests that next
generation sequencing technology will have a
transformative effect on public health control of M.
tuberculosis despite being a tool in relative infancy.
There is no standardized approach to assembling
andfilteringrawdata,andnoconsensusaroundhow
results should be interpreted to guide public health
interventions.
Inferences drawn from genetic data depend on the
epidemiology of the setting, the completeness of
sampling, the sequencing platform and filtering
pipeline, as well as methods used to relate sequences
to one another. Whereas there are attempts to build
aninternationalconsensusaroundfilteringpipelines,
it is equally important to build an understanding of the
caveats when interpreting the outputs of any agreed
approach. I will use case studies to discuss the
reasons for caution when interpreting NGS data, as
well as to highlight the potential power of the tool.
L-3
ADVANCES IN MOLECULAR TYPING OF
MyCOBaCTERIUM TUBERCULOSIS; SHOULD
WHOLE GENOME SEqUENCING BECOME THE
NEW GOLD STANDARD?
Dick van Soolingen1, Josephine Bryant2, Rosa
Sloot3, Martien Borgdorff4, Jessica De Beer5
1
national Institute for public health and the
Environment, Bilthoven, netherlands
2
Wellcome Trust Sanger Institute, Cambridge, UK
3
Department of Clinical Epidemiology,
Biostatistics and Bioinformatics, academic
Medical Center, University of amsterdam,
amsterdam, The netherlands
4
Department of Infectious Diseases, public
health Service, amsterdam, The netherlands
5
national Tuberculosis Reference Laboratory,
Environment (Rivm), Bilthoven, The netherlands
Mycobacterium tuberculosis (Mtbc) reveals a very
limited genomic diversity and does generally not
horizontally exchange DNA, which suggests whole
genome sequencing (WGS) may be an excellent tool in
the molecular epidemiology. However, this requires a
particular rate of genetic turn-over, ideally in agreement
with the pace of tuberculosis (TB) transmission. To
study this, 199 isolates from epidemiologically linked
or unrelated cases with known time spacing were
selected at the Municipal Health Service in Amsterdam
and subjected to WGS. An average mutation rate of
~0.3 single nucleotide polymorphisms (SNPs) per
genome per year was observed. However, there
was a very high degree of variation in mutation rate
around this mean. Furthermore, not less than 82 pairs
33
without an obvious epidemiological link revealed a
zero SNP difference, suggesting interpretation of
WGS data to detect epidemiological links may be
difficultinoursetting.However,alsoapartoftheepilinks indicated by RFLP/vNTR were refuted by the
information from WGS. Once the WGS technology
is capable of including repetitive sequences such
as the MIRu-vNTRs and IS6110 elements in the
analysis,thiswillbecomeahighlyefficientlaboratory
toolforidentification,indicativeresistancetestingand
epidemiological typing.
L-4
BENCH TOP NExT GENERATION SEqUENCING:
REVOLUTIONIZING MyCOBaCTERIUM
TUBERCULOSIS MOLECULAR EPIDEMIOLOGY
AND DIAGNOSTICS?
Stefan Niemann
Molecular Mycobacteriology, Research Center
Borstel, Borstel, germany
Next generation sequencing (NGS) based whole
genome analysis (WGS) of clinical Mycobacterium
tuberculosis complex (MTBC) isolates opens the
door for completely new approaches for molecular
epidemiology and tuberculosis (TB) diagnostics.
Few pioneering studies indicate that WGS offers
substantially higher resolution of MTBC clinical strains
e.g. for outbreak investigation compared to classical
genotyping such as IS6110 typing or 24 locusbased MIRu-vNTR typing. Simultaneously, WGS
analysis allows for almost comprehensive detection
of variants in target genes involved in resistance
development (resistome analysis), thus potentially
outcompeting classical molecular diagnostic tests
that can determine a limited number of variations
(mainly single nucleotide polymorphisms, SNPs) in
few targets only.
So far, real time prospective NGS based analysis
of clinical isolates was hampered by the fact that
NGS analysis have been mainly performed by
specialized sequencing centres in a retrospective
manner on large expensive NGS platforms that are,
intermsofworkflow,sequencingcostsandruntime,
not optimized to the needs of smaller laboratories
investigating bacterial genomes. However, recently,
so-called bench top NGS systems, which can be
integrated into a normal laboratory workflow, have
become available to sequence bacterial genomes in
few days.
We established rapid genome analysis of clinical
MTBC isolates using the Illumina MiSeq system
and software pipelines usable in a small laboratory
settings.Theoverallworkflowfromlibrarypreparation
tofinalNGSdataanalysiscouldbeoptimizedtolast
less than three working days. First evaluations of
the performance indicate that MiSeq based genome
analysis is excellently suited for real time investigation
of MTBC outbreaks and diagnostics.
34
ABSTRACTS OF GUEST LECTURES (GL)
GL-1
NEW INSIGHTS INTO PATHOGENOMICS OF THE
TUBERCULOSIS AGENT
Roland Brosch
Institut pasteur, Unit for Integrated Mycobacterial
pathogenomics, paris, France
Global spread and limited genetic variation are
hallmarks of M. tuberculosis, the agent of human
tuberculosis. In contrast, Mycobacterium canettii
and related tubercle bacilli that also cause human
tuberculosis and exhibit unusual smooth colony
morphology are restricted to East Africa. We
sequenced and analyzed the whole genomes of
five representative strains of smooth tubercle bacilli
(STB) using Sanger, 454/Roche and Illumina DNA
sequencing as well as four strains on the basis of
a shotgun assembly. We show that STB isolates
are highly recombinogenic and evolutionarily early
branching, with larger genome sizes, higher rates of
genetic variation, fewer molecular scars and distinct
CRISPR-Cas systems and prophages relative to M.
tuberculosis. Despite the differences, all tuberculosiscausing mycobacteria share a highly conserved core
genome. Mouse infection experiments showed that
STB strains are less persistent and virulent than
M. tuberculosis. We conclude that M. tuberculosis
emerged from an ancestral STB-like pool of
mycobacteria by gain of persistence and virulence
mechanisms.
Apart from genomic regions that differed, we also
found regions that were highly conserved among
the different strains. One of these regions was the
genomic locus encoding the ESX-1/type vII secretion
system. This system, which is absent from the
attenuated BCG and Mycobacterium microti vaccine
strains, has been recently shown to be involved
in the rupture of the phagolysosomal membrane in
THP1 cells. The presence of ESX-1 or its absence
from certain mycobacterial strains or strain-lineages
thus strongly influences the virulence potential
and the immunological properties of a given strain.
This knowledge is of importance for the search of
potential virulence factors of M. tuberculosis and for
the construction of better recombinant vaccines and
diagnostic tools.
GL-2
MOLECULAR EPIDEMIOLOGY OF
TUBERCULOSIS SPREADING IN EUROPEAN
METROPOLITAN AREAS
Molecular epidemiology of MDRTB
strainsTBPANNET Working Group
Andrea Gori
Division of Infectious Diseases, „San gerardo“
hospital, University of Milano-Bicocca, Monza,
Italy
Background: The study aims to characterize the
molecular epidemiology features of drug resistant TB
strains in selected metropolitan settings in Western
and Eastern Europe creating new tools to monitor
the spreading of drug-resistant TB as well as to
study social, epidemiological and clinical risk factors
associated with TB in different urban settings.
Patients and Methods: Consecutive collection of
all M. tuberculosis strains isolated and of clinical and
epidemiological data in order to perform a systematic
study of the risk factors for MDR-TB. Standardization of
genotyping data reporting and interpretation between
different metropolitan settings and development of an
easy-to-use TB surveillance system for the automated
identificationofM. tuberculosis strain types.
Results: 2520 more strains were collected in
2012. Cluster analysis of 360 MDR-TB strains with
complete 24-loci MIRU-VNTR profile isolated in 7
centres, demonstrated a clustering rate of 0.647
using 24-loci MIRu-vNTR genotyping. Rate of MDR
TB clusterization varied across centres (from 95.4%
in Tartu, 68.9% in vilnius, 53.6% in Bruxelles, 38.5%
in Stockholm, 19.4% in London). The most common
lineage of MDR-TB strains was Beijing (65.6%),
followed by uRAL (11.1%) and LAM (7.2%). Other
lineages included Haarlem (1.7%), Delhi/CAS (1.1%),
TuR (0.8%), Cameroon (0.8%), Ghana (0.6%), NEW1 (0.3%), ugandaI (0.3%). The largest MDR-TB
cluster included 148 strains belonging to the Beijing
family, of whom 130 isolated in Tartu, 13 in vilnius, 4
in Bruxelles and 1 in Hamburg. Local-born patients
weresignificantlymorelikelytobeinvolvedinclusters
than foreign-born patients in the cities of Hamburg
(44.3% clustered patients among local-born versus
22.2% among foreigners; P=0.008), London (20.7%
versus 6.8%; P=0.014), vilnius (53.8% versus 36.8%;
P=0.15) and Tartu (70.5% versus 56.8%, P<0.001).
Conversely, the risk of clustering was comparable
between local- and foreign-born patients diagnosed
with TB in Stockholm and Bruxelles. Homelessness
(OR 1.51; 95%CI 0.97-2.36), recent detention (OR
2.6; 95%CI 1.69-4.00), intravenous drug abuse (OR
35
1.89; 95%CI 0.96-3.72) and alcohol abuse (OR 2.12;
95%CI 1.65-2.74) were associated with a higher risk
of clustering. Moreover, HIv-positivity was associated
with a higher risk of clusterization as compared
with patients with a negative test (OR 2.76; 95%CI
1.27-6.02). No evidence of increased risk of MDRTB clustering was found among prisoners, alcohol
abusers, intravenous drug users and patients using
immune-suppressive drugs.
Conclusions: A wide approach integrating molecular
dataandtheepidemiologicalinformationtodefineTB
transmission settings represents an helpful method in
designing and in improving health public preventive
strategies, and in elaborating new tools to control the
spreading of MDR-TB in the setting of Western and
Eastern European metropolitan cities.
direct WGS for all specimens. Taking WGS to predict
multiple resistance profiles for XDR-TB to more
challenging diagnostic environments will require novel
nano-sensingtechnologiesandmicrofluidicsthatare
currently under development. Genotypic correlates
of resistance have the potential for major impact in
clinical management of XDR-TB and indirectly will
benefit public health as well as provide major cost
benefits to healthcare systems. Capacity for and
access to accurate and rapid phenotypic antibiotic
susceptibility testing remains imperative and will allow
for continual robust genotypic correlates of resistance
to be made. Dedicated curated and interrogatable
databases are needed. TB clinicians will need to use
the language of genomics, as has been achieved in
HIv medicine.
GL-3
GL-4
RAPID WHOLE GENOME SEqUENCING AS A
TOOL TO MANAGE MDR/xDR-TB PATIENTS
LESSONS ON DRUG RESISTANCE FROM WGS
OF ONE THOUSAND CLINICAL ISOLATES
Philip D Butcher
Centre for Infection and Immunity, St george’s
University of London, London, UK
Taane G Clark (on behalf of the Global Drug
Resistance Collaboration)
Faculty of Infectious and Tropical Diseases,
London School of hygiene and Tropical
Medicine, London, UK
Genome sequencing has had a transformative
impact on our understanding of the pathogenesis
and the biology of TB. But what clinical impact has
it had? The W.H.O. calls for better control measures
if the millennium goal of TB eradication by 2050 is
tobemet.Efficientclinicalcasemanagementwillbe
a major contributor, but new technologies to inform
better diagnostics and treatment are also called for in
addition to new vaccines and new drugs. Genomics
can contribute to these. One area of potential
impact is the use of whole genome sequencing
(WGS) to predict antibiotic susceptibility from
genotypic changes in target genes by sequencing
clinical isolates of M.tuberculosis.This is of specific
relevance to the management of MDR and XDR TB,
where cocktails of antibiotics are given for prolonged
periods, often empirically. Rapid access to genotypic
data could inform treatment regimes. Several
examples are presented of how WGS has been used
in different clinical cases of XDR-TB. These include
theidentificationofsinglenucleotidepolymorphisms
(SNPs) in gyrA to predict the use of moxifloxacin
despite the DST predicting resistance; and SNPs in
pncA that informed the exclusion of pyrazinamide from
a treatment regime. Consideration of how this can
be routinely implemented in hospital care pathways
requires WGS data to be rapidly available within days,
without the need for culture and direct from sputum
specimens. Turn around times of 2 days are possible
using the IonTorrent PGM sequencer. Methods to
differentially enrich M.tuberculosis DNA from human
sputumarerequiredsothatsufficientsequenceread
depth can be obtained for robust SNP calling from low
amounts of bacterial DNA. Consideration of variation
in bacterial load in sputum will affect the applicability of
36
The increase in incidence of multi- and extensivedrug resistance (MDR, XDR) is a serious challenge
to the effective control of tuberculosis. There is
an urgent need for better treatments and further
insights into drug resistance mechanisms, which in
turn require a deeper understanding of the biology
of TB. Knowledge of the genomic variability among
Mycobacterium tuberculosis (Mtb) could result in
such biological insights. using recent innovations
in massively parallelisable sequencing technology,
we have whole genome sequenced over 1000
Mtb (sourced from 10 countries) with known drug
resistance profiles (MDR, XDR) and strain-types.
We present results and insights from an ongoing
genomewide association analysis.
GL-5
LOW DENSITY MICROARRAYS AS POTENTIAL
TOOLS FOR THE DIAGNOSIS OF DRUG
RESISTANT TUBERCULOSIS
Paolo Miotto
Emerging Bacterial pathogens Unit
Division of Immunology, Transplantation and
Infectious Diseases
San Raffaele Scientific Institute Milan, Italy
The lack of diagnostic tools and drugs is the main
obstacle to control poverty-related diseases.
Tuberculosis (TB), malaria and AIDS account for 15-
20% of the disease burden in the poorest countries.
The diagnosis of TB is challenging due to the
complexity of its clinical presentation. In addition, the
emergence of drug resistant (DR) strains represents
an additional obstacle to prompt clinical management
of TB cases. Performing sensitivity tests in solid or
liquid media is still a challenge in many settings due
to the inadequacy of infrastructures, the suboptimal
biosafety and the long time required to obtain results.
For some drugs, including first line drugs, problems
of reproducibility and accuracy of drug susceptibility
testing (DST) cast an additional layer of entanglement.
The WHO estimates that less than 4-6% of suspects
are tested for multidrug resistance (MDR) DST
globally, and only 23% of MDR cases were reported
to have second-line DST. The suboptimal levels of
coverage of DST contributes to the low diagnosis of
MDR-TB cases.
Todayseveralmutationsinwell-definedregionsofthe
genome of M. tuberculosis (MTB) are associated with
DR phenotype and can be targeted for a molecular
DST. The advent of such tests, mainly based on
the detection of the rifampicine resistance, greatly
boosted the detection of MDR cases. However, soon
after their introduction in the diagnostic algorithms
it became clear that further resistance testing is
needed for a successful management of the patients.
Moreover, several evidences shows the clinical
relevance of the genetic background of MTB and
support the need to introduce genotyping data (usually
used only for epidemiological purposes) in the clinical
routine. To address this demand for an extended DST
evaluating multiple genetic targets, low/mediumthroughput tools came into play. Microarrays have
the unprecedented potential to simultaneously detect
and identify several microbial genes and/or provide
parallel sequencing analysis. For these applications,
microarray platforms are undergoing an adaptation
to switch from translational research laboratories to
clinical laboratory. Costly devices requiring extensive
expertise are being replaced by user-friendly labon-chips integrating different steps and providing
automated analysis and reporting. As PCR and
real-time PCR have done in the past, microarray
technology will undoubtedly transform the diagnostic
capabilities of clinical laboratories.
GL-6
MOLECULAR AND qUANTITATIVE APPROACHES
TO DST – TOOLS TOWARDS BETTER GUIDED TB
THERAPY IN THE DAYS OF MDR AND xDR
Erik Christian Boettger
Institut für Medizinische Mikrobiologie,
nationales zentrum für Mykobakterien,
Universität zürich, zürich, Switzerland
insight that has accumulated during the past years,
little has changed in the laboratory procedures which
define drug resistance for clinical M. tuberculosis
isolates on the basis of critical concentration testing.
Evidence is accumulating that drug resistance in M.
tuberculosis is quite heterogeneous and composed
of low-, moderate-, and high-level drug resistance.
Systematic genotype – phenotype studies have
revealed that different SNPs are associated with
different resistance levels. These findings indicate
that established procedures for diagnostic drug
susceptibility testing based on critical concentration
testing have limitations and need to be complemented
by quantitative measures of drug susceptibility, with
the view to optimize treatment in particular for MDR/
XDR tuberculosis.
GL-7
CHALLENGES OF PYRAZINAMIDE TESTING
Sven Hoffner
The Swedish Institute for Communicable Disease
Control, Solna, Sweden
Pyrazinamid(PZA)isanimportantfirstlineagentin
the treatment of tuberculosis (TB), with a clear role
in both drug susceptible cases and in patients with
drug resistant TB, as long as the infecting strain is
susceptible to PZA. unfortunately in most MDR/
XDR-TB cases the PZA susceptibility is not known
since information of in vitro susceptibility to PZA, at
least reliable information, is lacking in most setting
where MDR/XDR-TB is common. The reasons for this
unfortunatedifferencetootherfirstlineanti-TBdrugs
are the well known technical difficulties to establish
reliable, high quality phenotypic susceptibility testing
for PZA, the lack of generally available proficiency
test panels and the lack of a commercially available
molecular rapid test to demonstrated resistance
related mutations in the pncA gene. The lack of quality
support for PZA DST is especially unlucky since PZA
testing is technically more demanding than any other
important anti-TB drug – and of course the fact that
we see increasing public health treats due to drug
resistant TB. Resistant M. tuberculosis strains need
to be treated with the best possible combinations of
anti-TB drugs.
In this presentation, our experience with the phenotypic
assays; MGIT and PZA:ase-test, sequencing of
the pncA gene as well as lessons learned while
introducing a national qMS for PZA DST in Sweden
will be discussed.
Acquired drug resistance in M. tuberculosis is
exclusively due to chromosomal alterations, such as
mutations or deletions. Compared to all the genetic
37
GL-8
GL-10
NEW APPROACHES TO TUBERCULOSIS
TREATMENT
NEW TB VACCINES, WHERE WE ARE AND
WHERE WE GO
Christian Lienhardt
Carlos Martin
GL-9
HOST-BASED BIOMARKERS AND DIFFERENT
OUTCOMES OF MTB INFECTION
Gerhard Walzl
Immunology Research group, DST/nRF Centre
of Excellence for Biomedical Tuberculosis
Research and MRC Centre for Molecular and
Cellular Biology, Division of Molecular Biology
and human genetics, Faculty of Medicine and
health Sciences, Stellenbosch University, Cape
Town, South africa.
Our incomplete understanding of host responses
during the different stages of Mycobacterium
tuberculosis (MTB) infection hampers the discovery of
new tools (affordable, rapid and accurate diagnostics,
effective treatment regimens lasting only a few weeks
and pre- and post exposure vaccines) to control
tuberculosis. We have searched for biomarkers for
different infection phases and for different outcomes
of infection and treatment in a high MTB transmission
setting with high TB disease rates in Cape Town, South
Africa, by using a combination of targeted host marker
measurements and unbiased ‘omics‘ approaches.
During active disease, disturbances in numerous
biological pathways are mirrored by changing levels
ofhostinflammatoryandanti-inflammatorymolecules
and recover at different rates during chemotherapy.
High serum levels of inflammatory host markers
and extensive disease at baseline predict slow
MTB culture conversion, relapse after initial cure or
treatment failure. Marked geographical differences
in immune responses within Africa were noted. It is
therefore unlikely that single biomarkers will succeed
as predictors of outcome but biosignatures that are
composed of several host markers look promising. We
also present the largest positron emission tomography/
computerized tomography (PET/CT) dataset in relation
to TB chemotherapy available to date, which reveals
remarkable end-of-treatment heterogeneity amongst
patients.Ongoinglunginflammationinalargesubset
ofpatientsmaybeduetosecondaryinflammationor
due to persistent MTB infection, in spite of negative
sputum cultures. In conclusion, although the search
for clinically useful TB biomarkers is ongoing, new
information is being generated in this search, which
increases our understanding of host responses to
MTB infection.
38
GL-11
NEW CHALLENGES FOR BURULI ULCER
CONTROL
Francoise Portaels, K. Vandelannoote, M.
Eddyani, B. de Jong
Mycobacteriology Unit, Institute of Tropical
Medicine, antwerp, Belgium
Buruli ulcer (Bu), caused by Mycobacterium ulcerans,
is the third most common human mycobacteriosis
worldwide after tuberculosis and leprosy. M.
ulcerans arose from M. marinum and acquired
a virulence plasmid coding for mycolactone, a
necrotizing, immunosuppressive toxin that mediates
pathogenesis.
The disease is endemic in rural wetlands of tropical
countries of Africa, America, Asia and Australia but
remains uncommon in non-African countries.
A few cases have been reported in non-tropical areas
of Australia, Japan and China. Some patients, who
were born and lived in Bu endemic countries or
traveled to endemic countries, developed Bu in a
non-endemic country.
These cases have enabled us to acquire more
knowledge regarding the pathogenesis of the disease
(latency and reactivation).
Children 15 years old or younger account for
approximately 75% of cases. Risk factors include
tropical climate, exposure to stagnant water,
unprotected water sources, hygiene, trauma to skin
and HIv infection.
The most plausible mode of transmission is minor
skin trauma permitting inoculation of M. ulcerans, but
other modes and reservoirs, are under investigation.
Geographically, there are multiple strains of M. ulcerans,
with variable pathogenicity and immunogenicity.
Fingerprinting techniques and genome sequencing
can provide additional information on the precise
location where patients were infected.
M. ulcerans disease presents a spectrum of forms
related partly to patient delay in admission to hospital
and other factors such as the immune status of the
host, the size and depth of the inoculum, and the
virulence of the M. ulcerans strain.
The clinical diagnosis can be difficult even for
experienced health professionals, hence the
importance of microbiologic confirmation by direct
smear examination, in vitro cultivation, PCR targeting
IS2404 by gel-based PCR or quantitative realtime qPCR and histopathologic examination (also
important for the differential diagnosis).
The results of new diagnostic methods such as
antigen capture approach and direct detection of
mycolactone are encouraging and are likely to lead to
atestthatcouldbeusedinfield.Theylack,however,
sufficientsensitivity.
WHO recommends, if not contraindicated, the directly
observed use of at least an 8-week course of rifampicin
and streptomycin (RS). Surgery is recommended to
remove necrotic tissue, cover large skin defects and
correct deformities. Combination of antibiotics and
surgery may be necessary for some severe lesions
such as disseminated and bone lesions.
Other antibiotic regimens, especially those
administered completely orally (rifampicin and
clarithromycin) are under evaluation in Benin and
Ghana.
Regarding possible vaccination, the Burulivac project
hasledtomanynewfindingsbutaneffectivevaccine
will not be available for many years.
GL-12
INTERFERON GAMMA RELEASE
ADVANTAGES, LIMITATIONS, NEW
ADVANCES
ASSAYS:
Delia Goletti
Translation Research Unit,
Department of Epidemiology and preclinical
Research national Institute for Infectious
Diseases, Rome, Italy
A third of the world’s population is estimated to be
infected with Mycobacterium tuberculosis, providing
a very large reservoir for future active tuberculosis
(TB). The tuberculin skin test (TST) has traditionally
been used to identify people with latent tuberculosis
infection (LTBI). Despite its usefulness and simplicity,
the TST has limitations, mainly due to logistic and low
specificityinthosevaccinatedwithBacillusCalmette
et Guerin. T-cell-based interferon (IFN)-γ release
assays (IGRAs) can also be used for the diagnosis
of LTBI and have been available for the past decade.
They are in vitro assays based on the overnight
releaseofIFN-γfromTcellsinresponsetospecific
antigens from the RD1 region of M. tuberculosis.
Here we discuss their accuracy in terms of sensitivity,
specificity,positiveandnegativepredictivevaluefor
TB development in both, immune competent and
immune deficient persons. Advances of IGRAs will
be presented. The new tests are based on antigens
different from RD1, on readout different from the
detectionofIFN-γ,onincubationtimedifferentfrom
overnight stimulation. Advantages and limitations will
be discussed.
39
ABSTRACT of
HONOREE Lecture
GERTRUD MEISSNER
AWARD LECTURE
Tuberculosis: new drugs on the
horizon
Causes and consequences of parallel
evolution of tuberculosis and humans
over seventy thousand years
Giovanna Riccardi
Department of Biology and Biotechnology
“Lazzaro Spallanzani”, University of Pavia, Pavia,
Italy.
Tuberculosis (TB) is still one of the most difficult
infections to treat. The prevalence of TB/HIV coinfection, and the emergence of multidrug-resistant
(MDR-TB), extensively drug-resistant (XDR-TB)
and even totally drug resistant (TDR-TB) strains of
Mycobacterium tuberculosis constitute a serious
health problem. Consequently, new antitubercular
drugs are urgently needed with novel mechanisms of
action.
Indeed, after a long period of neglect, a few molecules
are now in phase II clinical trials while others are still
in preclinical phase, like the benzothiazinone BTZ043.
This is one of the most promising TB drug candidate
which targets the catalytic component of the essential
enzyme
decaprenylphosphoryl-β-D-ribofuranose2‘-epimerase (DprE1), involved in the biosynthesis
of arabinans, crucial components of mycobacterial
cell wall and essential for the pathogen’s survival.
The recent description of the crystal structures of M.
tuberculosis DprE1 enzyme in native form and bound
with BTZ will contribute to a better understanding of
the inhibition mechanism as well as to design new
and more appropriate molecules against this “magic
target”.
There is currently no consensus on the best method
to identify new anti-TB drugs that may be effective
in vivo. A simple approach is to discover an active
molecule through a phenotypic screening of chemical
or natural libraries against M. tuberculosis in vitro
growth, and then to identify the corresponding target.
An alternative way includes first the identification
of new essential mycobacterial targets and then
a suitable inhibitor, being aware that compounds
identified by this screen often lack activity against
whole cells. It is noteworthy that in the battle against
TB, all of the molecules currently under development
or in clinical trials were discovered according to their
whole cell activity.
New drugs, as well as the corresponding targets,
identified in our laboratory, through the selection of
spontaneous resistant mutants followed by whole
genome sequencing, will be reported.
40
Iñaki Comas1, Mireia Coscolla2, Tao Luo3,
Stefan Berg4, Dorothy Yeboah-Manu5, Sebastien
Gagneux2, Julian Parkhill6, Qian Gao3, Douglas
Young7, Stefan Niemann8
1
Center for Public Health Research (CsispFisabio), Valencia, Spain
2
Department of Medical Parasitology and
Infection Biology, Swiss Tropical and Public
Health Institute, Basel, Switzerland
3
Key Laboratory of Medical Molecular Virology,
Institutes of Biomedical Sciences and Institute
of Medical Microbiology, Fudan University,
Shanghai, China
4
Tb Research Group, Veterinary Laboratories
Agency, Weybridge, New Haw, Addlestone,
Surrey, UK
5
Noguchi Memorial Institute for Medical
Research, University of Ghana, Legon, Ghana
6
Pathogen Genomics, The Wellcome Trust
Sanger Institute, Wellcome Trust Genome
Campus, Hinxton, Cambridge, UK
7
Mrc National Institute for Medical Research, Mill
Hill, London, UK
8
Resarch Center Borstel, National Reference
Center for Mycobacteria, Forschungszentrum
Borstel, Borstel, Germany
Tuberculosis (TB) caused 20% of all human deaths in
the Western world between the 17th and 19th centuries,
and remains a cause of high mortality in developing
countries. In analogy to other crowd diseases, the
origin of human TB has been associated with the
Neolithic Demographic Transition, but recent studies
point to a much earlier origin. Here we used 259 wholegenome sequences to reconstruct the evolutionary
history of the Mycobacterium tuberculosis complex
(MTBC). Coalescent analyses indicate that MTBC
emerged about 70 thousand years ago, accompanied
migrations of anatomically modern humans out of
Africa, and expanded as a consequence of increases
in human population density during the Neolithic. This
long co-evolutionary history is consistent with MTBC
displaying characteristics indicative of adaptation
to both low- and high host densities. During this
seventy thousand years tuberculosis strains have
accumulated more than thirty thousand single
nucleotide polymorphism. I will show examples how
this long-term co-evolution has translated in genetic
changes that impact the physiology of the bacteria
and the nature of the host pathogen interaction
ABSTRACTS OF ORAL PRESENTATIONS (OP)
M. TUBERCULOSIS GENOMICS I
OP39
IDENTIFICATION OF LINEAGE-SPECIFIC GENETIC MARKERS FROM MORE THAN 1,500 MTBC
ISOLATES
Francesc Coll1, Kim Mallard1, Mark D. Preston1,
Stephen Bentley2, Julian Parkhill2, Ruth McNerney1, Nigel Martin3, Taane G Clark1
1
Faculty of Infectious and Tropical Diseases,
London School of hygiene and Tropical Medicine, London, UK
2
Wellcome Trust Sanger Institute, Wellcome
Trust genome Campus, hinxton, UK
3
Department of Computer Science and Information Systems, Birkbeck College, London, UK
Single nucleotide polymorphisms (SNPs) and large
sequence polymorphisms (LSP) derived from wholegenome sequencing (WGS) are becoming the markers of choice for epidemiological and evolutionary applications, replacing those from traditional genotyping
techniques such as IS6110-RFLP, spoligotyping or
MIRu-vNTR. To take full advantage of the high volumes of raw data being generated by current WGS
technologies, we have developed a computational
pipeline to discovery genetic polymorphisms. SNPs,
small insertions and deletions and LSPs have been
catalogued for more than 1,500 MTBC isolates using state-of-the-art sequence analyses tools. The
variants have been used to construct phylogenetic
trees, showing clear population structure across major MTBC genetic lineages including both modern
and ancestral lineages. Isolates were in silico spoligotyped to investigate strain diversity, and population
genetics metrics were applied to identify strain specificvariantsandthosedrivinglineage.Theresulting
informative markers will assist the development of
high-throughput assays to barcode MTBC isolates for
epidemiological, diagnostic and clinical studies. Further, we have developed a web-based tool to display
the resulting MTBC variation in the global dataset
integrated with allele geographic distribution, strain
type information and population structure visualisation through the construction of phylogenetic trees
OP157
COMPARATIVE TRANSCRIPTOMIC ANALYSIS
OF MyCOBaCTERIUM TUBERCULOSIS IN A
LIPID ENVIRONMENT
Patricia Del Portillo1, Maria Jesus Garcia2, Jorge
Gonzalez-y-Merchand3, Maria Mercedes Zambrano1, Juan Germán Rodríguez1, Juan Manuel
Anzola1
1
Corporación Corpogen, Bogota, Colombia
2
Facultad de Medicina, Universidad autónoma de
Madrid, Madrid, Spain
3
national School of Biological Sciences, Ipn.,
Intituto politecnico nacional, Mexico City, Mexico
Introduction: Emerging evidence suggest that
fatty acids rather than carbohydrates might be the
dominant carbon substrate utilized by Mycobacterium
tuberculosis (Mtb) during intracellular growth and
persistence. Mtb is considered to be in a dormant
stage during latency where bacilli reside in a lipid
environment provided by the surrounding foamy
macrophages. To determine the role of fatty acids
in the metabolism and persistence of Mtb, we used
ss-RNA-seq to analyze changes in the transcriptome
of Mtb associated with the assimilation of even-long
chain fatty acids (LC-FA) as sole carbon source.
Methods: For the in vitro model, bacilli were grown
in either dextrose-supplemented (control) or in a mix
of LC-FA. Total RNA was isolated in the exponential
and early stationary phases of growth and directional
libraries were constructed. Sequencing was performed
using Illumina HiSeq platform. Statistical analysis for
differential gene expression was performed using
the Fisher Exact Test with False Discovery Rate
correction.
Results: The number of sequences retained after
processing for quality was 13.1 – 19.3 million reads,
the libraries showed a good coverage of the Mtb
genome with the sequence depth obtained. The global
results showed that in the presence of fatty acids,
the number of transcripts were lower. Interestingly,
regulatory RNAs, are more transcribed and are
differentially expressed in the stationary phase of
fatty acid enriched environment. The overexpression
of aminoacyl-t-RNAs was observed for the first
time. Several genes belonging to different functional
categoriesshowedsignificantincreaseexpressionin
the fatty acid model. Among them those related with
lipid metabolism allow us to propose a novel model
that will be discussed.
Conclusions: The results show the potential of this
technology to give a holistic view of the transcriptome
of MTB under selected conditions that in the future
could contribute towards the design of novel strategies
for the control of tuberculosis.
Funding resources: The FP7 Eu-grant 200999
- Stop LATENT-TB and Colciencias Grant No 4392012 funded this work
41
OP53
INSIGHTS INTO THE GENE ACTIVITY OF MyCOBaCTERIUM TUBERCULOSIS GROWING IN A
FATTY-ACID ENRICHED MEDIUM
Juan German Rodriguez1, Adriana Carolina
Hernandez1, Jorge Gonzalez-y-Merchand2, Addy
Cecilia Helguera-Repetto2, Jose Ricardo Bustos1,
Juan Manuel Anzola1, Maria Mercedes Zambrano1, Patricia del Portillo1, Maria Jesus Garcia3
1
2
Encb, Instituto politecnico nacional, Mexico
3
Introduction: In order to survive during infection,
Mycobacterium tuberculosis (MTB) should confront
different environmental conditions. Thus, resistance to
oxidative or acidic stresses are considered pivotal for
its subsistence. Besides, there is cumulative evidence
that host lipids may be a main energy source for MTB
to establish a successful infection. The accumulation
of lipids by the bacilli is particularly relevant during
their intracellular growth, such increase of these
compounds leads to a considered state of persistence
or dormancy.
Objectives: The main objective of the work was
to unravel the transcriptomic response of MTB in a
lipid environment. We focus on the analysis of the
transcriptomic activity emphasizing the functional
categories of the genes expressed under such
conditions.
Methods: Total RNA was isolated in the exponential
and early stationary phases of growth from MTB
cultures in both dextrose and long-chain fatty acidenriched media (FA-EM). Results derived from global
transcriptomic sequencing (RNAseq) were analyzed
by standard functional categories of the genes. We
sought to identify genes with significant changes in
expression in fatty-acids versus dextrose using the
Fisher exact test.
Results: We identified genes with significant
increased expression in the exponential and stationary
phases of the FA-EM cultures when compared
with corresponding phases in the dextrose media.
Therefore, these genes characterized the adaptation
of the bacilli to growth in a FA-EM and thus provided
a mycobacterial lipid-signature, which includes genes
involved in different biological functions of Mtb.
The lipid signature includes genes members of five
functional categories as described in Tuberculist.
As described previously, our results also showed
that the glyoxylate shunt was the preferred metabolic
pathway for energy production and Acetyl-CoA
recycling, being the Icl and PckA the pivotal enzymes.
Some genes that codes for protein secretion systems
appears to be putatively selected for use in lipid
transportation. In addition, we detected up to four
regulatory proteins with significant expression on
FA-EM, including WhiB3 and DosR, two regulators
related to dormancy. Remarkably, almost half of the
42
genes in the lipid signature were members of the
dormancy regulon.
Conclusions: We suggest the mycobacterial growth
in FA-EM as a novel in vitro model suitable to study
the dormant phase of MTB.
Funding resources: This work was funded by the
FP7 Eu-grant StoplatEnt-tB.
OP246
GENERATION AND CHARACTERIZATION OF A
MyCOBaCTERIUM TUBERCULOSIS DELETION
MUTANT DEFICIENT IN A PUTATIVE SUBUNIT OF
A HETERODIMERIC ABC MULTIDRUG TRANSPORTER
Sven Malm, Silvia Maaß, Stefan Ehlers, Stefan
Niemann
Research Center Borstel, Leibniz-Center for
Medicine and Biosciences, Borstel, germany
Mycobacterium tuberculosis remains a major health
threat for the world’s population and still had been
the cause of 1.4 million deaths in 2011. The outcome
of tuberculosis is balanced to a considerable degree
by the interplay of exposed mycobacterial molecules
such as cell wall constituents with the host’s immune
system. The translocation of cell wall constituents
and their precursors is crucial for exposition of these
molecules on the bacterial surface as well as for
completion of their synthesis on the extracellular
site of the plasma membrane and a lack of mature
molecules on the mycobacterial surface might
lead to attenuation of virulence or loss of cell wall
rigidity. In Escherichia coli MsbA is a transporter
of the membrane associated cell wall component
lipid A, a constituent of LPS. MsbA is a lipid A ATPdependent transmembrane transporter with flippase
activity. msba of E. coli is an essential gene, however
mutations within msba can lead to a relaxed substrate
specificity.MsbAhastwohomologueswithunknown
function in M. tuberculosis, therefore here referred to
as MsbA1TB and MsbA2TB. It is proposed that msba1TB
and msba2TB encode ABC multidrug transporters.
Most probably these ORFs originated from a gene
duplication event and are organized in an operon. Both
genes might encode a heterodimeric transport protein.
A transposon mutant of msba1TB has been found to
be essential in vivo and attenuated in macrophages.
Transposon mutational analysis of msba2TB has
shown that this gene is essential for the survival in
macrophages.Aimofthisworkistocontributetofind
the link between the intra- and extracellular synthesis
of glycolipids of M. tuberculosis, in particular PIMs and
theirhighermolecularderivativesandalsotodefine
its significance for virulence and pathogenesis in
mycobacterial infection. Here we report the generation
of a M. tuberculosis deletion mutant deficient in
msba2TB. Initial characterization of the mutant strain
revealed increased sensitivity of the mutant strain to
osmotic stress suggesting that the mutation affected
either cell wall integrity or osmoregulation.
OP164
CHARACTERIZATION OF THE SMALL RNA ExPRESSION PROFILES INDUCED BY ANTIBIOTICS IN MyCOBaCTERIUM TUBERCULOSIS
Paolo Miotto1, Matthias Merker2, Barbara Tizzano2, Davide Cittaro3, Dejan Lazarevic3, Elia
Stupka3, Stefan Niemann2, Daniela Maria Cirillo1
1
Emerging Bacterial pathogens Unit, Division
of Immunology, Transplantation and Infectious
Diseases, San Raffaele Scientific Institute, Milan,
Italy
2
3
Center for Translational genomics and Bioinformatics (Ctgb), San Raffaele Scientific Institute;
Milan, Italy
Small non-coding RNAs (sRNAs) play an important
role in the stress response and the pathogenicity of
many bacteria.
This study aims to characterize the expression
profileofsRNAsincombinationtomessengerRNAs
(mRNAs) in Mycobacterium tuberculosis (MTB) under
stress conditions induced by antibiotic exposure.
M. tuberculosis H37Rv was grown in Middlebrook
7H9 medium containing 0.05% Tween80 and
supplemented with 10% ADC until early exponential
phase.Weexposedtheculturetoofloxacin(OFX)and
moxifloxacinantibioticsatbacteriostaticconcentration.
Mitomycin C (MMC) was used as additional stressor
inducing genotoxic stress. Total RNA was extracted
at different timepoints (T0, T1 = 30 min, T2 = 180 min)
during antibiotic treatment and checked for quality and
quantity by Bioanalyzer 2100 (Agilent Technologies).
Libraries for strand-specific sequencing of mRNAs
were prepared according to the Ovation RNA-Seq
System (NuGEN) protocol or to the TruSeq Small
RNA sample preparation guide (Illumina) for sRNAs.
In both cases, libraries were run on HiSeq 2000
(Illumina). Transcriptome analysis was performed
by aligning the reads to the reference genome using
bwa. Strand specific counts of previously published
sRNA species were normalized and analyzed using
R/BioConductor packages edgeR and maSigPro, in
order to identify sRNAs with differential pattern of
expression in time.
OFX challenge showed a time-dependent response
and 4 sRNA clusters (namely c1, c2, c3, and c4)
significantlymodulatedduringdrugchallenge.Major
changes were observed at T1. Expression levels for
2 of clusters (c1, c3) were found to be consistent to
those observed in MMC treated MTB. The c2 showed
minor changes between OFX or MMC treated and
control cultures. The remaining sRNA gene cluster
43
showed to be up-regulated during MMC treatment
but slightly down-regulated during OFX challenge,
compared to untreated bacteria.
Our preliminary data suggest an “early time response”
during OFX stress. We identified a small subset of
sRNAs showing differential expression between OFX
and MMC treatment. Integrating sRNA and mRNA
expression data from different stress experiments
will allow the development of post-transcriptional
regulatory networks providing novel insight into the
fundamental biology of tuberculosis.
OP23
UNDERSTANDING THE BIOLOGY OF POLYRIFAMPICIN RESISTANCE IN MyCOBaCTERIUM
TUBERCULOSIS
Margaretha de Vos1, Gail Erika Louw2, Rob Warren1, Thomas Victor1, Paul van Helden1
1
Stellenbosch University, Faculty of Medicine
and health Sciences, Tygerberg, South africa
2
Tuberculosis Research Section, Lcid/niaid/nih,
Bethesda, USa
Background: Previous studies have shown that
rifampicin Minimum Inhibitory Concentrations (MIC’s)
are highly variable in in vitro selected rifampicin
resistant isolates harbouring the same rpoB
mutation. This led to the question whether a similar
phenomenon occurred in vivo. However, population
heterogeneity would not be seen during routine MIC
determination as only clones with the highest level of
resistance within a culture will be measured. Thus,
MIC determination assays masks the true population
structure of resistance phenotypes with a bacterial
culture. Determination of the rifampicin MIC’s of single
Colony Forming units (CFu) isolated from sputum
cultures showed a spectrum of MIC’s irrespective of
the rpoB mutation and strain background (similar to the
in vitro experiments). This in turn led to the question
as to whether the observed phenotypic heterogeneity
was heritable. Subsequent MIC determination after
subculture of the original CFU confirmed, in all
instances, that the rifampicin MIC phenotype was
stably inherited.
Aim: To use whole genome sequencing and
bioinformatics tools to identify genetic differences
(SNPs, In/Dels) which define the level of rifampicin
resistance in isogenic rifampicin-mono-resistant M.
tuberculosis clones extracted from sputum cultures.
Materials and methods: CFU’sreflectingtheextreme
MIC’s (i.e. 2µg/ml vs >100µg/ml) for each rifampicinmono-resistant sputum culture (n=14) were cultured
on 7H10 media and DNA was extracted. The whole
genomes of 41 CFu‘s were sequenced using an
Illumina platform. Comparative bioinformatics tools
were used to identify genetic differences between
the CFu‘s selected from the same progenitor. This
included the use of different sequence aligners
and variant callers to maximize the identification of
44
high confidence variants. H37Rv was used as the
reference genome.
Results and Discussion: An initial analysis was
conducted on three CFu‘s (R721_C13, rifampicin
MIC: 2 µg/ml; R721_C1, rifampicin MIC: 70µg/ml;
R721_C14: rifampicin MIC: 100 µg/ml). Comparative
analysis between these CFu‘s showed that R721_C1
and R721_C14 are genetically similar, while four
putative In/Dels (small insertions and deletions)
were identified in clone R721_C13. No SNPs were
detected between the three clones. These In/Dels will
be validated by Sanger sequencing and additional
bioinformatics tools will be used to predict the effect of
the In/Dels on protein function. Additionally, analysis
of the remaining CFu‘s will reveal whether a common
mechanism is used to modulate the level of rifampicin
resistance.
Conclusion: Identificationofmechanismmodulating
the levels of rifampicin resistance may be more
complex than the accumulation of SNPs. This
study will address unique knowledge gaps in our
understanding of the biology of drug resistance.
OP260
WHOLE GENOME SEqUENCING FROM EARLY
CULTURE OR DIRECTLY FROM CLINICAL
SAMPLES
Mathilde Mairey1, Bénédicte Condamine1,
Christine Hubans-Pierlot1, Stéphanie Ferreira1,
Philip Supply 2, Caroline Allix-Béguec1
1
genoscreen, Lille, France
2
Genoscreen, Lille; INSERM, U1019, CNRS UMR
8204, Institut pasteur de Lille, Univ Lille nord de
France, Lille, France
Background: Whole genome sequencing (WGS)
has the potential to become the ultimate method for
molecular epidemiology1. However, one key limitation
of current WGS technologies for medical microbiology
applications is the minimal amount of genomic DNA
necessary for the library preparation (1 ng to 1 µg
according to platforms).
Objectives: For slow growing species like
Mycobacterium tuberculosis, first-line diagnostic
screening relies on microscopy with a detection limit
of 104 bacilli per milliliter which corresponds to around
0.1 ng of genomic DNA. The detection limit of gold
standard culture methods is even lower. Therefore,
there is a need to optimize the sample preparation
process in order to be able to use genomic DNA
extracted from early culture or directly from clinical
samples.
Methods: We have assessed whole genome
amplification (WGA) strategies in order to increase
the amount of genomic DNA. Therefore, we have
designed and conducted experiments to evaluate
the potential of relevant and commercially available
WGA kits with respect to 6 key parameters: DNA
enrichmentrate,amplificationreproducibility,minimal
locus bias and sequence error rate, compatibility with
subsequent WGS analysis, and price per test.
Conclusion: One kit gave reproducible WGA results,
whichallowedustoamplifygenomicDNAinsufficient
quantity. It also required the shortest experiment and
hands-on times, while retaining a competitive price
per test. More importantly, subsequent WGS analysis
indicated low sequence error rates. The detected
SNPsandIndelsarenowsubjectedtoverification.
Reference: 1 Roetzer et al. (2013). PLoS Med 10(2):
e1001387. doi:10.1371/journal.pmed.1001387
OP245
GENOME WIDE ASSOCIATION ANALYSIS OF
TUBERCULOSIS RESISTANCE IN DAIRY CATTLE
Mairead Bermingham1, Steve Bishop1, John
Woolliams1, Adrian Allen2, Stewart McBride2,
David Wright2, Jon Ryder2, Robin Skuce2, Stanley
McDowell2, Liz Glass1
1
Roslin Institute, University of Edinburgh, Midlothian, Scotland, UK
2
agri-Food and Biosciences Institute, veterinary
Sciences Division, Belfast, UK
Mycobacterium bovis is the aetiological agent of
bovine tuberculosis (TB). Diagnosis is based on the
tuberculin test and abattoir surveillance. The failure
of the uK and several countries to eradicate TB
indicates a need to consider alternative strategies,
such as genetic selection of cattle for increased
resistance to TB. The aim of this study was to conduct
a case/control genome wide association study using
the newly available Illumina bovine high density SNP
chip to identify loci associated with variation in TB
resistance in the Northern Ireland Holstein-Friesian
dairy cattle population. Blood samples from 3,715
cattle were collected from 464 herds between 2008
and 2009. Cases were sampled at slaughter, and
weredefinedasanimalswithbothapositivereaction
tothetuberculinskintestandaconfirmedTBlesion.
Age matched controls were sampled from a subset of
case herds, and were defined as animals that were
negative to the tuberculin skin test. In total, 1,424
cattle were genotyped for 777,962 SNPs. After qC
edits, 1,151 cattle (592 cases and 559 controls)
and genotype data from 617,610 SNPs remained.
Genome wide association using mixed models and
regression was used to test for associations between
SNPs and TB resistance. The estimated heritability
for resistance to TB was 0.21 (SE 0.06). Chromosome
wide significant associations detected at eight loci,
and together these SNPs explained 8.2% of variance
in resistance to TB in the cattle population sample
investigated in this study. We aim to independently
replicate these findings and understand the genetic
architectureofthesignificantregions.
45
OP47
SUCCESSFUL RUSSIAN CLONE OF MyCOBaCTERIUM TUBERCULOSIS: ORIGIN, EMERGENCE
AND CURRENT SPREAD
Igor Mokrousov
St. petersburg pasteur Institute, St. petersburg,
Russia
Background and objective: It is very common yet
true saying that M. tuberculosis is a major human
killer. However some of its lineages and clones show
more capacity to spread and cause active disease. A
clonal group named B0 (Narvskaya, 2003) or W148
(Bifani et al., 2002) was reported in 1/4 of the Beijing
genotype isolates across former Soviet union and
in respective immigrant communities in the uSA
and Western Europe. It has been hypothesized
that Beijing В0/W148 historically recently spread
throughout former Soviet union due to its special
pathogenic properties and thus may present a
successful clone of M. tuberculosis (Mokrousov et
al., 2008). However, an integral picture of its origin,
evolutionary history and biological properties remains
obscure. Did it originate within the borders of Russia
or former Soviet union or was imported, for example
from East Asia? What human movement served,
unwittingly, as a vehicle of its dissemination? What
pathobiological properties underlie its spread? I
searched the literature for presence and properties
of the Beijing В0/W148 strains in local populations;
these data were subjected to systematic and critical
review, re-analysis and new analyses.
Review and analysis: Genetically, Beijing B0/W148
is marked with a characteristic double band (7.1 and
9.2 kb) in the upper part of the IS6110-RFLPprofile.In
St. Petersburg, most of these isolates had 24-MuRu
profile100-32(MIRU-VNTRplus).Althoughthisprofile
100-32 is prevalent among B0/W148 isolates in St.
Petersburg,noconsensus24-MIRUprofileexistsfor
B0/W148 isolates across Russia. Geographically, in
spite of the common perception about omnipresence
of Beijing B0/W148 across all post-Soviet countries,
its distribution shows a peculiar clinal gradient. Its
frequency peaks in the Siberian Russia and, to a
lesser extent, in the European part of the former
Soviet union. In contrast, the rate of B0/W148 is
sharply decreased in the Asian part of the former
Soviet union and it is absent in the autochthonous
populations elsewhere in the world. Placing the
molecular, clinical and epidemiological features in the
broad historical, demographic and ecological context,
I put forward two interdependent hypotheses. First,
46
B0/W148 likely originated in Siberia and its primary
dispersalwasdrivenbyamassivepopulationoutflow
from Siberia to European Russia in the 1960-80s.
Second, a historically recent and phylogenetically
demonstrated successful dissemination of the Beijing
B0/W148 strain was triggered by an advent and
wide use of the modern anti-TB chemotherapy and
was due to its remarkable capacity to acquire drug
resistance. In contrast, there is some indication, but
not yet a systematic proof, of the enhanced virulence
of this strain. Future studies on more isolates from
new locations and new time points (archival samples)
will increase density of data and resolution of analysis
and will test these hypotheses.
OP73
LARGE SEqUENCE POLYMORPHISMS OF LATIN
AMERICAN-MEDITERRANEAN (LAM) MyCOBaCTERIUM TUBERCULOSIS STRAINS ISOLATED
IN ITALY AND MOLECULAR EPIDEMIOLOGY OF
THE LAM RDRIO STRAINS
Dipartimento di Ricerca Traslazionale e delle
nuove Tecnologie in Medicina e Chirurgia, Università Di pisa, pisa, Italy
The Latin American-Mediterranean (LAM) lineage of
the Mycobacterium tuberculosis complex constitutes a
widespread family of strains within the Euro-American
phylogeographical lineage. In this study we report
the genotypic characterization, based on the large
sequence deletion and spoligotype polymorphisms, of
a collection of LAM strains isolated in Tuscany, Italy, a
region with a low prevalence of tuberculosis (TB), but
where the ethnic diversity of TB patients provides an
opportunity to study a global sample of LAM strains.
Large sequence polymorphism (LSP) analysis of a
collection of 137 LAM strains detected three prevalent,
mutually exclusive deletions, i.e., RD115, RD174 and
RD726, respectively in 27.7%, 28.5% and 13.9% of
isolates; 94.9% of strains bearing deletion RD174
also harboured the RDRio deletion. Deletions RD182,
RD219 and RD761 were detected in occasional
strains; deletions RD122, RD183, RD193 and RD724
were not found; 24.1% of strains showed no deletion.
Deletion RD726 was found highly prevalent in the
LAM10_CAM spoligotype sublineage that includes
the “Cameroon” strains. Of the 42 Spoligotype
International Types (SITs) assigned to the study
strains, eleven were shared by strains belonging to
differentdeletion-definedsublineages.Inparticular,9
SITs (SIT20, 33, 61, 93, 161, 177, 209, 737 and 1064)
were detected in two RD-defined sulineages; 1 SIT
(SIT60) in three sublineages; and 1 SIT (SIT42) in
as many as six distinct RD sublineages. The general
lack of concordance between the phylogenetic
sublineagesdefinedbylargesequencedeletionsand
the spoligotype groupings provides a clear evidence
of spoligotype homoplasy due to independent deletion
events of the same spacers in strains belonging to
different evolutive lineages. These considerations
argue against the use of spoligotyping for defining
deep phylogenetic relationships within the LAM
family.
With regard to the LAM RDRio strains, recently emerged
as an important cause of tuberculosis (TB) associated
with a more severe disease, high transmissibility and
multidrug resistance, we did not observe any clinically
distinctive or more severe form of disease in RDRio TB
patients; moreover, 15-loci variable-Number-TandemRepeat (vNTR) analysis demonstrated that the very
large majority of RDRio strains show unique genotypic
profiles,whichrulesoutanincreasedtransmissibility
of RDRio strains in our low-incidence setting.
networks and the underlying expansion of successful
clones. Furthermore, our data indicate that a small
number of SNPs can lead to altered fitness and
increased spreading of a particular clone. Finally, we
deduced the Mtb mutation rate in its natural host and
the maximum level of genome variation in humanto-human transmissions. Our results establish a
paradigm for WGS based pathogen tracing which
offers the unique opportunity to unravel the turnover in
time and space of pathogenic strain variants in human
populations, allowing for a fine-scale estimation of
theirrelativefitnessandadaptivemutations.
OP224
TRACING MyCOBaCTERIUM TUBERCULOSIS
TRANSMISSION: WHEN RESOLUTION MATTERS
Thomas Kohl1, Roland Diel2, Stefan Niemann1
1
2
Institute for Epidemiology, Schleswig-holstein
University hospital, Campus Kiel, germany
Reliable and highly discriminatory genotyping of
Mycobacterium tuberculosis strains (causative
agents of tuberculosis, TB) is essential for early
outbreak detection and the characterization of
transmission chains, thus greatly improving TB
control strategies. As classical genotyping (e.g.
based on IS6110 DNA fingerprint or 24-loci-MIRUvNTR typing) targets only a tiny part of the genome,
it may not be able to distinguish between closely
related transmission chains, especially over longer
time frames . Furthermore, little is known about
the microevolutionary events that affect pathogen
transmission and fitness in human populations. In
contrast to classical genotyping, whole genome
sequencing opens new opportunities for tracing the
spread of pathogens and exploring genome evolution
with the highest possible resolution. However, its
performance and public health relevance for tracking
outbreaks and genome evolution has not been
elucidated.
To address this question, we performed whole genome
sequencing (WGS) of more than 140 strains from
nine different outbreaks found by classical genotyping
from 1997 to 2011 in the City of Hamburg. Overall,
our results shows WGS analysis to be superior to
classical genotyping for the analysis of transmission
47
OP43
DETECTION OF RIFAMPICIN RESISTANCE
IN MyCOBaCTERIUM TUBERCULOSIS
BY PADLOCK PROBES AND A MAGNETIC
NANOBEAD-BASED READOUT
Anna Engström1, Teresa Zardán Gómez De La
Torre2, Maria Strømme2, Mats Nilsson3, David
Herthnek4
1
Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden;
Department of preparedness, Swedish Institute
for Communicable Disease Control, Solna, Sweden
2
Department of Engineering Sciences, Division
of nanotechnology and Functional
Materials, Uppsala University, The Ångström
Laboratory, Uppsala, Sweden
3
Science for Life Laboratory, Department of
Biochemistry and Biophysics,
Stockholm University, Stockholm, Sweden
4
Department of Immunology, genetics and
pathology, Uppsala University, Science for
Life Laboratory, Rudbeck Laboratory, Uppsala,
Sweden
Background: Drug resistance poses great
challenges for the prevention, treatment and control
of tuberculosis (TB). In order to quickly identify a
drug-resistant strain it is essential to use molecular
diagnostic methods, which can be performed within a
day. Padlock probes (PLPs) are linear oligonucleotides
with target complementary regions at their 5’ and 3’
ends. The PLP ends are brought into juxtaposition
upon hybridization to the target sequence, allowing
PLP circularization by ligation. A mismatch at the
3’ end hinders ligation, thereby providing a specific
means of mutation detection.
Methods: PLPs were designed to target the most
common mutations associated with rifampicin (RIF)
resistance in Mycobacterium tuberculosis, i.e. at
codons 516, 526 and 531 in rpoB. For detection
of the wild type sequence, a modified PLP and
two oligonucleotides were used, requiring three
ligation events (at the above mentioned codons) for
circularization. Circularized probes were amplified
by rolling circle amplification (RCA). RCA products
were
coupled
to
oligonucleotide-conjugated
magnetic nanobeads and detected by measurement
of the Brownian relaxation frequency shift in an AC
susceptometer.
Results: The PLPs were tested for discriminatory
power on RIF-susceptible and RIF-resistant strains
harboring a wild type or mutated rpoB gene. The
sensitivity was determined to 30 ng of M. tuberculosis
DNA in the magnetic bead-based detection assay.
48
The method proved to be robust for discrimination of
specificmutationsandconfirmationofmaintainedor
lostwildtype,andforidentificationofM. tuberculosis
complex DNA.
Conclusions: We have used PLPs, RCA and a
magnetic biosensor readout format to develop a
molecular method for detection of RIF resistance in
M. tuberculosis. The magnetic biosensor is suitable
for future development of a low-cost diagnostic labon-a-chip device.
OP89
SPRINT: SPOLIGO-RIFAMPICIN-ISONIAZID
TYPING
Michel Gomgnimbou1, Ivan Hernández-Neuta2,
Stefan Panaiotov3, Elizabeta Bachiyska3, Palomino Juan Carlos4, Anandi Martin5, Patricia Del
Portillo6, Guislaine Refrégier7, Christophe Sola7
1
Université paris-Sud-Cnrs, Umr8621, Institut de
génétique Et Microbiologie, the Infection, genetics, Emerging pathogens (Igepe) Team, Orsay,
France; Centre Muraz, Bobo-Dioulasso, Burkina
Faso
2
Molecular Biotechnology Corporacion Corpogen, Bogota, Colombia
3
national Center of Infectious and parasitic Diseases, Sofia, Bulgaria
4
University of gent, gent, Belgium
5
Laboratory of Microbiology, Department of Biochemistry and Microbiology, ghent University,
gent, Belgium
6
7
Université paris-Sud-Cnrs, Institut de génétique
Et Microbiologie, Infection genetics Emerging
pathogen Evolution Team, Orsay, France
Multi and Extensively Drug-Resistant tuberculosis (M/
XDR-TB) is a threat for public health. It needs urgent
improvement in the field of diagnostic, treatment,
control and prevention. We extended previous
results on “spoligoriftyping” that allows simultaneous
spoligotyping and rifampicin resistance mutations
detection on Mycobacterium tuberculosis complex
(MTC) DNA, to the detection of isoniazid resistance
mutations. This work introduces a new paradigm
for tuberculosis control by putting a microbiology
laboratory as a hub to provide simultaneously, on
one hand genotypic Drug-Susceptibility Testing
(DST) results to clinicians for patient treatment
management, and on the other hand data for molecular
epidemiology purposes to epidemiologists and public
health specialists. Targeted mutations in the rpoB,
katG, inha, and the DR locus are simultaneously
amplified by PCR. Hybridization detection is done
optically in a microbead-based assay on a Luminex
200®, BioPlex®, FlexMap 3D® or on a Magpix® system.
We called this test “SPRINT” and we validated the
test using sequencing as reference, phenotypic
DST and spoligotyping. Spoligotyping patterns
obtained by “SPRINT” were 100% (n= 85 isolates;
3,655/3,655 spoligotype data points) concordant to
those previously obtained by spoligoriftyping and
membrane-based method. Compared to sequencing,
the “SPRINT” method was 100% concordant for MTC
(n =162 for rpoB gene sequencing and n =76 for
katG and inhA sequencing) rifampicin and isoniazid
resistance associated mutations detection (targeted
mutations). Considering the phenotypic DST as
referencetest,thesensitivity/specificityof“SPRINT”
regarding MTBC (n= 162 isolates) rifampicin and
isoniazid resistance were respectively 100% /100%
and 90.4% (CI95% = 85-95%) /100%. used routinely
in national reference laboratories and where culture
is available, the “SPRINT” test should improve TB
diagnostic, patients’ personalized treatment and
allow real-time epidemiological investigations and
monitoring. This method could play an increasingly
important role for public health in the countries with
an heavy burden of multi-drug resistant tuberculosis
and/or HIv/TB co-infection. Progress towards fast
XDR-TB detection using the same principles are in
progress together to the use of “SPRINT” directly on
clinical specimen.
OP154
DEVELOPMENT OF AN In vITRO SYSTEM TO
PERFORM TIME-KILL CURVES OF LEVOFLOxACIN AGAINST MyCOBaCTERIUM TUBERCULOSIS
Aline Barth1, Robert May2, Judith Johnson2,
Charles Peloquin1, Hartmut Derendorf 1
1
College of pharmacy – University of Florida,
gainesville, USa
2
College of Medicine – University of Florida,
gainesville, USa
Multidrug-resistant TB present resistance to the
most effective drugs and are treated with second line
drugs (SLDs) that are more toxic and less effective.
To evaluate the causes of the reduced effectiveness
of levofloxacin (LEV), a main SLD, we developed
an in vitro system that expose the microorganism to
differentdrugconcentrationprofiles.Ouraimistouse
this system to investigate the pharmacodynamics of
LEv using the “dynamic time-kill curves”.
Methods: In this model, a peristaltic pump
continuously pumps fresh sterile broth into the main
flask containing the microorganism, and a second
pump removes the broth. A magnetic stir bar ensures
homogeneity of the culture and prevents membrane
pore blockage.
The initial validation of the system was done with a
single dose of LEv equivalent to the human1000mg
dose administered in Middlebrook 7H9 broth and
samples were collected over 24 hours. The objective
was to verify if the human elimination profile could
be mimicked. We also performed a 10 days study
to evaluate the growth of the H37Ra strain (ATCC
25177) and the use of bovine calf serum (CS) instead
of ADC as enrichment for the liquid broth, in order to
reduce costs. After that, a control study was done over
the course of 7 days using the system with no drug.
A sample was drawn daily and serial dilutions were
performed to calculate CFu/ml and growth curve. The
MIC for the LEv was then established using 2 fold
dilution.
Results: TheeliminationprofileofLEVwasproperly
emulated, as constant of elimination of 0.08h-1 was
obtained (literature value 0.09h-1).The use of CS as
a broth supplement provided reduced growth, and
thereforeADCwasfurtherused.Thegrowthprofileof
the microorganism within the system was obtained.
The LEv MIC was established as 0.75 µg/mL.
Conclusion: Our system was appropriately
developed and it is capable of mimicking the human
drug concentration profile. The attenuated strain
is initially being used to ensure the safety. In future
trials, we intend to use clinical strains. Our next steps
are to use different drug doses that will correspond
to multiples of the MIC values (0.5, 1 and 16 MIC) in
order to create a mathematical model that correlates
the pharmacodynamics with the pharmacokinetics,
allowing to simulate and predict the LEv concentrations
that optimize treatment, and to define the related
doses. using this information it will be possible to
simulate the effects of real world circumstances, i.e.
missed doses, in vitro. The difference between this
system and other techniques is the ability to change
the environment in a controlled setting to monitor how
the organism responds to different stimuli.
OP212
RAPID TESTING FOR DRUG SUSCEPTIBILITY
Antonino Catanzaro1, Timothy Rodwell1, Camilla
Rodrigues2, Valeriu Crudu3, Thomas Victor4, Kanchan Ajbani2, Elena Romancenco5, Andre Trollip4,
Cindy Hayes6, Roberta Lynn Jackson1, Theodore
Ganiats1, Erik Groessl1, Richard Garfein1
1
University of California, San Diego, USa
2
p.D. hinduja national hospital and Medical Research Center, Mumbai, India
3
Center for health policies and Studies (pas Center), Chisinau, Moldova
4
Stellenbosch University, Faculty of health Sciences Stellenbosch University, Tygerberg, South
africa
5
The Institute of phthisiopneumology, Chisinau,
Moldova
6
national health Laboratory Service, Johannesburg, South africa
49
Background: Standard phenotypic drug susceptibility
testing (DST) takes up to 3 months to identify XDRTB. The Global Consortium for Drug-Resistant
TB Diagnostics (GCDD; funded by NIH Grant
#5U01AI082229) was formed to develop and study
rapid XDR-TB diagnostics in >1000 subjects at three
clinical sites in Mumbai (India), Chisinau (Moldova)
and Port Elizabeth (South Africa). Primary study aims
include reducing XDR-TB detection time to a week
and determining agreement between rapid tests and
standard DST. Preliminary results are presented.
Methods: TB patients with risk factors for drugresistance were recruited from 3 multinational sites.
We performed AFB smear, MGIT960 culture and
DST, pyrosequencing (PSQ), line probe assay (LPA)
and MODS assays. We evaluated resistance to the
TB drugs isoniazid (INH), rifampin (RIF), moxifloxacin
(MOX), ofloxacin (OFX), amikacin (AMK), capreomycin
(CAP) and kanamycin (KAN).
Results: Preliminary results based on samples from
~650 subjects collected between 2012 and 2013
are presented. Preliminary data for the MGIT DST
indicated a median time-to-result (TTR) of 25 days
(n=484). Median TTR for the MODS assay was 15
days (n=442), while PSQ was only 8 days (n=606)
and Hain LPA (plus and sl) was 5 days (n=641).
Rapid test accuracy was compared to MGIT960 DST
as the Gold Standard. For the MODS test (N=288290) INH sensitivity and specificity were 95% and
99%; RIF sensitivity and specificity were 99% & 95%;
MOX sensitivity and specificity were 98% & 97%;OFX
sensitivity and specificity were 98% & 98%. AMK
sensitivity and specificity were 86% & 100%; CAP
sensitivity and specificity were 95% & 99%; KAN
sensitivity and specificity were 48% & 100%. For the
PSQ assay (N= 346-360) INH sensitivity and specificity
were 96% & 96%; RIF sensitivity and specificity were
97% & 98%; MOX sensitivity and specificity were 95%
& 96%; and OFX sensitivity and specificity were 95%
& 98%. AMK sensitivity and specificity were 86% &
99%; CAP sensitivity and specificity were 85% & 99%;
KAN sensitivity and specificity were 48% & 99%. For
the Hain MTBDRplus LPA (N=399-409) INH sensitivity
and specificity were 95% & 99%; RIF sensitivity and
specificity were 98% & 94%. For the Hain MTBDRsl
LPA (N=334-360) MOX sensitivity and specificity were
95% & 97% and OFX sensitivity and specificity were
95% & 98%. AMK sensitivity and specificity were 79%
& 100%; CAP sensitivity and specificity were 85%
& 99%; KAN sensitivity and specificity were 40% &
100%. Concordance with MGIT DST was MODS: INH
96%; RIF 98%; MOX/OFX 98%; AMK and CAP 99%;
KAN 91%. PSQ: INH 96%; RIF 97%; MOX 95% and
OFX 96%; AMK and CAP 98%; KAN 92%; Hain LPA:
INH 96%; RIF 97%; AMK and CAP 98%; KAN 92%;
MOX 96% and OFX 97%.
Conclusions: Our preliminary data indicate that rapid
diagnostics reduced XDR-TB diagnosis time from
months to days. Rapid results were highly sensitive
and specific for INH, RIF and FQ resistance, but
showed <90% sensitivity for injectable drugs.
50
OP229
KANAMYCIN, AMIKACIN, CAPREOMYCIN
CROSS RESISTANCE IN MyCOBaCTERIUM TUBERCULOSIS ISOLATES FROM ROMANIA
Daniela Homorodean1, Andrea Melinda Jodal1,
Sven Hoffner2
1
Clinical hospital of pneumology Leon Daniello,
national Reference, Laboratory, Cluj napoca,
Romania
2
The Swedish Institute for Communicable Disease Control, Solna, Supranational Reference
Laboratory Stockholm, Sweden
The cross resistance between Kanamycin (KM),
amikacin (AMK), capreomycin (CAP) among
Mycobacterium tuberculosis strains is a subject of
debate in the last years.
In the first drug resistance survey of second line
anti-tuberculosis drugs resistance among multidrug
resistant tuberculosis (MDR-TB) patients in Romania
(2009-2010), kanamycin resistant (KMr) strains and
kanamycin-ofloxacin resistant strains prevalences
were 27.8% and 11.4% respectively. CAP was not
used in Romania before this survey.
We aimed to assess the level of cross resistance
between KM, AMK and CAP among Mycobacterium
tuberculosis strains isolated from MDR-TB patients.
Material and methods. For external quality control
of the survey results, 72 randomly selected strains
out of 756 clinical MDR isolates, were retested in
Supranational TB Reference Laboratory in Sweden
for the drugs tested in the survey (isoniazid, rifampicin,
KMandofloxacin)andalsoforAMKandCAP.
Results. Seventeen out of 72 (23.6%) strains were
susceptible to KM, AMK, CAP; 34 (47.2%) strains
were resistant to all this drugs; 7 (9.7%) strains were
resistant to CAP and one aminoglycosid (KM in 2, and
AMK in 5 cases). Five strains were resistant to one
aminoglycosid but susceptible to CAP. Nine (12.5%)
strains were resistant to only CAP.
In ten out of 50 (20%) CAP resistant strains, the
corresponding patients were not treated before with
any aminoglycoside (KM, AMK, or streptomycin), and
we consider these cases as initial CAPr.
Conclusions. These results have diagnostic and
therapeutic implications: a. Strains isolated from all
MDR-TB patients should be tested for KM, AMK and
CAP if all these agents are considered for therapy;
b. Aminoglycosides should be used in therapeutic
regimens of MDR-TB cases only after in vitro
susceptibility test (genetic or phenotypic) results are
available.
OP243
EPIDEMIOLOGICAL TRENDS IN TB AND M/xDRTB AND PROGRESS ON THE IMPLEMENTATION
OF THE CONSOLIDATED ACTION PLAN TO
PREVENT AND COMBAT M/xDR-TB IN THE WHO
EUROPEAN REGION
Kristin Kremer, Andrei Dadu, Martin van den
Boom, Pierpaolo de Colombani, Masoud Dara
Who Regional Office for Europe, Tuberculosis
and M/XDR-TB programme, Copenhagen, Denmark
In 2011, 295,968 out of the estimated 380,000 new
TB cases were reported in the European Region and
more than 44,000 deaths were attributed to TB. Since
2007, TB notification has been decreasing in the
Region by an average of 5% per year. Among newly
reported TB cases, the number of MDR-TB cases
increased steadily from 4% in 2005 to 14% in 2011.
Of an estimated 78,000 MDR-TB cases, about 30,000
(38%) were detected in 2011, with the large majority
(98%) reported in the 18 high TB priority countries.
The prevalence of MDR-TB among previously treated
TB patients was 47.7% in 2011. The HIv prevalence
among incident TB cases increased from 2.8% in
2006 to 6.4% in 2011.
Since the endorsement of the Consolidated Action Plan
to Prevent and Combat Multidrug- and Extensively
Drug-Resistant Tuberculosis (M/XDR-TB) in the
WHO European Region 2011–2015 and the adoption
of its accompanying resolution by the WHO Regional
Committee for Europe in September 2011, most of the
milestones outlined in the action plan have been met.
Key achievements include the establishment of the
European Green Light Committee (GLC), along with
the provision of state of the art technical assistance
to Member States on MDR-TB, and the launch of
the European TB Laboratory Initiative (ELI) to scale
up quality diagnosis. Task forces have also been
established to update Member States’ knowledge of
childhood TB, to consider the role of surgery in TB,
to develop a consensus document on cross-border
TB control and care, and to assess and address the
health system and social determinants of TB in line
with Health 2020.
Despite having tripled since the endorsement of
the Action Plan, coverage of second line drug
susceptibility testing (DST) among MDR-TB cases
tested for second line drugs is still only 12%. The DST
results showed that up to 11% of all MDR-TB cases
are extensively drug resistant.
Treatment coverage for MDR-TB patients increased
from 63% of estimated MDR-TB patients in 2011,
to 96% in 2013, however, treatment outcome is
51
below the 75% target (48.5%) because of the lack
ofefficientmedicines,poorprogrammeperformance
and inadequate patient-centred approaches, as well
as the lack of a mechanism for cross-border TB care.
Of the 15 countries with a high MDR-TB burden, nine
had achieved universal access to MDR-TB treatment
and care by January 2013. However, gaps persist in
some Member States with patients still on waiting lists
to receive treatment.
OP244
EMERGENCE OF xDR IN HIGH BURDEN MDRTB
COUNTRY: A THREAT TO PUBLIC HEALTH AND
TB CONTROL PROGRAM
Elena Romancenco1, Valeriu Crudu2, Ecaterina
Noroc3, Nadejda Turcanu3, Liliana Domente3,
Sofia Alexandru3
1
State Medical and pharmaceutical University,
Chisinau, Moldova
2
Center for health policies and Studies (pas Center); Phthsisiopneumology Institute, Chisinau,
Moldova
3
The Institute of phthisiopneumology, Chisinau,
Moldova
Background: Emergence of extensively drug-resistant tuberculosis (XDRTB) is a threat to public health
and National TB Control Program in high burden
MDRTB country. Tuberculosis resistance represents
a serious obstacle to effective TB control in Moldova.
The results of drug resistance surveillance (DRS) can
reflecttheindicatorsofTBcontrolprogrammeefficacy.MDR&XDRTBisoneofthemaincausesofineffective treatment of TB cases.
The aim of the study: to estimate the trends of TB drug
resistance in Moldova during the period of 2006-2011.
TheresultsofDRStofistandsecondlineTBdrugsamong
new and previously treated TB cases were studied.
Methods: DRS included all isolates from new patients diagnosed with pulmonary TB during the
last 7 years. The DST was performed in the National TB Reference Laboratory and three Regional
Tuberculosis
Reference
Laboratories.
Results: The prevalence of the TB resistance increased considerably during the last period. In the
performed study it was analysis results of DRS from
7240 TB New Cases and 6997 previously treated TB
cases. The level of primary drug resistance has been
increased from 42,0% to 49,2% during this period,
and MDR TB from 19,4% to 29,2%. Among previously treated patients the level of MDRTB increase from
50,8% up to 63,4%. Trough patients with MDRTB detectedin2011the13,8%wereidentifiedwithextensively drug resistance.
The level of XDRTB increases considerable during the last two years. In 2010-2011 the level of XDR among MDRTB patients was 10,0%
and 13,8% (in 2007- 6.9%). The highest resistance to second line TB drugs was detecting to
52
Kanamycin and Ofloxacin – 18,0% and 11,6%.
Conclusion: At the current stage the TB drug resistance is a serious problem in the R.Moldova, having
serious public health and economic consequences.
Theincreasenumberofresistantcasesinfluencesof
the treatment results. The accumulation of a greater
number of resistant strains in society can lead to the
infection of population and increase the number of
TB resistant cases. The additionally, new measured
need to be implemented urgently for stopped this
situation.
OP96
MyCOBaCTERIUM TUBERCULOSIS BEIJING
GENOTYPE STRAINS ARE CAPABLE OF RESISTING TRANSIENT ExPOSURE TO RIFAMPICIN, AS STUDIED IN A NOVEL MICROCOLONY
APPROACH
Alice den Hertog1, Sandra Menting1, Dick van
Soolingen2, Richard Anthony1
1
Kit Biomedical Research, Royal Tropical Institute, amsterdam, netherlands
2
national Institute for public health and the Environment, Bilthoven, Netherlands; Department
of Medical Microbiology, Radboud University
nijmegen Medical Centre, nijmegen, The netherlands
using a culture method developed in which individual
microcolonies are monitored over time and growing
colonies can be moved from one solid medium
to another, we investigated the effect of transient
exposure to RIF on colony growth rate and post
antibiotic outgrowth on a panel of tuberculosis strains
of the Beijing and East-African Indian (EAI) genotypes.
In total 36,408 colonies were investigated.
We have found that after exposure to 0.5, 1.0 or 2.0
mg/L RIF for 4 hours, the growth of colonies from
the non Beijing strains studied (2 EAI strains and
H37Ra) was almost completely inhibited during the
first5daysafterexposure.Incontrastthe6Beijing
genotype strains studied showed residual growth of
the majority of colonies monitored in this period. Thus,
the Beijing colonies were more capable of persisting
growth during exposure to low concentrations of RIF.
We believe that the persistent slow replication of the
Beijing strains in the presence of low concentrations
of RIF may allow the generation of mutants post
exposure in stressed cells.
These observations may help explain the association
of Beijing genotype with drug resistance and relapse,
as the drug levels achieved during treatment may
be much more critical with respect to preventing the
accumulation of RIF resistant mutants for these strains
than for other genotypes. The microcolony method
is highly promising for fast phenotypic resistance
testing.
OP46
ANTI-TUBERCULOSIS ACTIVITY OF EFFLUx
INHIBITORS AGAINST DRUG RESISTANT MyCOBaCTERIUM TUBERCULOSIS
Diana Machado1, Isabel Couto2, Jorge Ramos1,
Miguel Viveiros1
1
grupo de Micobactérias, Unidade de
Microbiologia Médica, Instituto de higiene e
Medicina Tropical, Universidade nova de Lisboa
(IhMT, UnL), Lisboa, portugal
2
Microbiologia Médica, Instituto de higiene
e Medicina Tropical, Universidade nova
de Lisboa (IHMT, UNL), Lisboa, Portugal;
Centro de Recursos Microbiológicos (CREM),
UnL, Faculdade de Ciências e Tecnologia,
Universidade nova de Lisboa, Quinta da Torre,
portugal
The major mechanisms associated with drug resistance
in M. tuberculosis involve mutations in target genes
and increased efflux. The aim of this study was to
investigate the in vitroactivityofeffluxinhibitors(EIs)
on the resistance levels of isoniazid (INH), evaluate
changes in expression of EPs genes upon exposure
to INH, and assessment of the bactericidal activity
of the compounds. We tested seven M. tuberculosis
strains:H37Rv,H37RvΔkatG, three multidrug resistant
strains, and two INH monoresistant strains. The EIs
assayed were verapamil, flupenthixol, haloperidol,
chlorpromazine and thioridazine. The strains were
characterized by standard antibiotic susceptibility
testing and INH semi-quantitative susceptibility
testing, in presence/absence of EIs, and genetic
analysisofspecificregionsofthekatG, inhA and rpoB
genes. The EIs ability to inhibit efflux was assayed
by a real-time semi-automated method that detects
the accumulation and extrusion of ethidium bromide
(EtBr). The bactericidal activity of the compounds was
investigated through time-kill studies. The expression
level of genes coding EPs (Rv1217c-Rv1218c,
mmpl7, Rv1258c, Rv1410c, efpA, mmr, Rv2459c
and pstB) was determined by qRT-PCR. The results
shown that i) INH resistance levels can be reduced
from high to low level in presence of EIs; ii) verapamil
was the EI most effective in reducing the resistance
toINH;iii)effluxofEtBrwasdetectedinallstrains;iv)
verapamil was the EI with the highest inhibitory effect
on real-time efflux of EtBr followed by flupenthixol,
chlorpromazine, thioridazine and haloperidol, v) timekill studies show that the compounds are bactericidal,
and vi) most of the EP genes tested were upregulated
in response to INH exposure. In this work, we showed
that inhibition of EPs by EIs lead to INH intracellular
accumulation and increased susceptibility to this
drug despite the presence of a mutation leading to
resistance. Our results show that INH seems to
act like an inducer that stimulates a general stress
response and the different levels of resistance to INH
are a balance between the induction of several EPs
that regulate the intracellular level of INH and the
mutation. The data presented evidences a possible
therapeutic value for compounds that have the
capacitytoinhibitmycobacterialeffluxpumpsviathe
retention of co-administered anti-mycobacterial drugs
thataresubjecttoefflux,suchasINH.
OP66
ORAL PHENYLBUTYRATE AND VITAMIN D3
INDUCE THE CATHELICIDIN LL-37 IN IMMUNE
CELLS: A DOSE FINDING STUDY FOR TREATMENT OF TUBERCULOSIS
Rubhana Raqib1, Akhirunnesa Mily1, Rokeya
Rekha2 S.M. Mostafa Kamal3, Protim Sarker2,
Zeaur Rahim1, Gudmundur Gudmundsson4,
Birgitta Agerberth5
1
International Centre for Dirrhoeal Disease
Research (ICDDR,B), Dhaka, Bangladesh
2
International Centre for Dirrhoeal Disease
Research (ICDDR,B), Dhaka, Bangladesh;
Department of Medical Biochemistry and
Biophysics, Karolinska Institutet, Stockholm,
Sweden
3
national Institute of the Diseases of the Chest
and hospital, Mohakhali, Dhaka, Bangladesh
4
Institute of Biology, University of Iceland,
Reykjavik, Iceland
5
Department of Medical Biochemistry and
Biophysics, Karolinska Institutet, Stockholm,
Sweden
Background: We have shown earlier that
4-phenylbutyrate (PB) can induce cathelicidin LL-37
expression synergistically with 1,25-dihydroxyvitamin
D3 in a lung epithelial cell line. We aim to assess
therapeutic doses of PB alone or in combination
with vitamin D3 for induction of LL-37 expression in
immune cells and enhancement of antimycobacterial
activity in monocyte-derived macrophages (MDM).
Methods: Healthy volunteers were enrolled in an
open trial. Three doses of PB [250 mg (Group-I), 500
mg (Group-II) or 1000 mg (Group-III)] twice daily (b.d.)
together with vitamin D3 {5000 Iu once daily (o.d.)},
PB (500mg b.d.) (Group-Iv) or vitamin D3 (5000 Iu
o.d.) (Group-v), were given orally for 4 days. Blood
was collected on day-0, day-4 and day-8. Plasma
was separated, MDM and non-adherent lymphocytes
53
were cultured. LL-37 transcript and peptide
concentrations were determined by qPCR and ELISA,
respectively. Concentration of 25-hydorxyvitamin D3
was determined by ELISA. MDM-mediated killing
of Mycobacterium tuberculosis (Mtb) (H37Rv) was
performed by conventional culture method.
Results: MDM from Group-II showed increased
concentration of LL-37 peptide and transcript at
day-4 while Group-I showed increased transcript at
day-4 and day-8 compared to day-0 (P<0.05). Both
Group-I and -II showed higher levels of transcript on
day-4 compared to Group-III and Group-v (P<0.035).
Lymphocytes from Group-II showed increased
induction of peptide on day-4 compared to Group-I
and Group-Iv (P<0.05), while Group-Iv showed
increased levels on day-8 compared to Group-I and
Group-III (P<0.04). Intracellular killing of Mtb on day4 was significantly increased compared to day-0 in
Group-I, -II and -v (P<0.05).
Conclusion: The results demonstrate that 500mg
b.d. PB with 5000 Iu o.d. vitamin D3 is the optimal
dose for the induction of LL-37 (peptide and transcript)
and intracellular killing of Mtb and this dose may have
potential application in the treatment of TB. This dose
is being used in a clinical trial of adults with active
pulmonary TB (NCT01580007).
OP225
STERILIZING ACTIVITY OF DRUG COMBINATIONS AGAINST ACTIVELY REPLICATING AND
NONREPLICATING MyCOBaCTERIUM TUBERCULOSIS
Giovanni Piccaro1, Federico Giannoni1, Donatella
Pietraforte2, Alessandro Mustazzolu1, Lanfranco
Fattorini1
1
Istituto Superiore di Sanità, Dipartimento Di
Malattie Infettive, parassitarie Ed Immunomediate, Rome, Italy
2
Istituto Superiore di Sanità, Dipartimento Di Biologia Cellulare e neuroscienze, Rome, Italy
Granulomas containing Mycobacterium tuberculosis
(Mtb) stages ranging from actively replicating (AR)
aerobic (A) bacilli to nonreplicating (NR) hypoxic (H)
bacilli coexist in the lungs of TB patients, thus it is
essential to find drug combinations killing AR+NR
cells. NR cells can be generated by the dormancy
Wayne model at pH=6.6, however this model may
not mimic the environment of active lesions where pH
is 5.5-6 or lower. To better mimic real conditions, we
evaluated the activity of 10 drugs including rifampin
(R), isoniazid (I), pyrazinamide (Z), ethambutol
(E), amikacin (AK), moxifloxacin (MX) and the
nitrocompounds PA-824 (PA), nitazoxanide (NZ),
niclosamide (NC), metronidazole (MZ), alone and in
combination, against AR bacilli (5-day-old cells: A5)
and NR bacilli (5-, 12-, 19-day-old cells: H5, H12,
H19),byamodifiedWaynemodelofMtbH37Rvat
pH=5.8. Mtb viability of 7, 14, 21 day drug-exposed
54
cells was measured by both CFu and a much more
sensitive method based on day-to-positivity (DTP)
in the MGIT 960 system (MGIT). A combination was
considered sterilizing if DTP was >100 days. In A
cultures, CFu and pH rapidly increased, while in H
cultures growth stopped and pH increased slightly.
Isoniazid, a control drug killing only AR cells, was
active against A5 (aerobic) cells, low active against
H5, slowly replicating (microaerophilic) cells, and
inactive against H12 and H19 (anaerobic) cells; MZ
was active only against H12 and H19 cells. MX and
AK were active against A5 and H5 cells while R, NZ,
PA were active against A5, H5, H12, H19 cells. To kill
all A+H cells, the 3-drug combination R+MX+AK was
potentiated by adding a fourth drug (PA, NZ, NC, MZ),
in comparison with R+I+Z+E, used for human therapy.
Among six R+MX+AK-containing combinations
tested, R+MX+AK+PA was the only one killing A5, H5,
H12, H19 cells in 14 days, as shown by lack of CFus
and of re-growth in MGIT (DTP >100 days) after drug
exposure. All other combinations including R+I+Z+E
killedall4populationsin≥21days.Toverifywhether
free radicals can be involved in R+MX+AK+PA
sterilization, reactive oxidizing species generated
by single drugs were determined by electron
paramagnetic resonance. In conclusion, we found a
new combination more potent than R+I+Z+E killing all
aerobes, microaerophiles, anaerobes and persisters
potentially living in granulomas. Overall, our Wayne
model of acidity+hypoxia should be considered when
studying activity of new drug combinations against
Mtb.
OP64
WHAT IS THE ROLE OF HORIZONTAL GENE
TRANSFER IN MYCOBACTERIAL DRUG RESISTANCE AND VIRULENCE?
Sylvia Cardoso Leão1, Katiane Santin1, Christiane
Lourenco Nogueira1, Paulo Jose Martins Bispo1,
Cristianne Kayoko Matsumoto2
1
Universidade Federal de São paulo, Escola
paulista de Medicina, São paulo, Brazil
2
Departamento de Microbiologia, Imunologia
e parasitologia, Universidade Federal de São
paulo, Escola paulista de Medicina, São paulo,
Brazil
Introduction: A nationwide outbreak of mycobacterial
surgical infections in Brazil (2004-2008) was caused
by a single strain of Mycobacterium abscessus subsp.
bolletii harbouring a ~60-kbp circular plasmid of the
broad-host-range IncP-1β subgroup. The plasmid,
pMAB01, was successfully transferred to Escherichia
coli by conjugation and transformation. Genomic
sequencing showed that pMAB01 contains a complete
system for conjugative DNA transfer and two genetic
load regions carrying antimicrobial resistance genes,
including the Tn5393c streptomycin-resistance
(str) transposon and a typical class 1 integron
with an incorporated gene cassette encoding an
aminoglycoside 6’-N-acetyltransferase (aac(6’)-Ib),
the dihydropteroate synthase type-1 gene (sul1),
and a conserved orf (orf5) that encodes a GCN5like N-acetyltransferase. Here we investigated the
contribution of this plasmid to drug resistance in M.
abscessus and E. coli.
Material and Methods: We evaluated the minimal
inhibitory concentration (MIC) of streptomycin
(STR), kanamycin (KAN), tobramycin (TOB), and
trimethoprim-sulfamethoxazole (SXT) against the M.
abscessus subsp. bolletii outbreak strain and E. coli
K12, harbouring or not pMAB01. The standard method
was performed according to CLSI guidelines.
Results and Discussion: MICs (µg/ml) of M.
abscessus subsp. bolletii harbouring pMAB01 were
>64 for STR, >64 for KAN, 32 for TOB and >32/608
for STX. MICs of the cured mycobacterium were 64
for STR, 8 for KAN, 16 for TOB and 8/152 for STX.
MICs of E. coli transformed with pMAB01 and E. coli
transconjugants were >64 for STR, >64 for KAN, 64 for
TOBand≤0.25/9.5forSTX.MICsofwildtypeE. coli
were4forSTR,2forKAN,1forTOBand≤0.25/9.5
for STX. In M. abscessus the plasmid contributed to
KAN resistance, but not to STR and TOB resistance.
MIC for STX was two doubling dilutions higher in
M. abscessus harbouring the plasmid than in M.
abscessus lacking it, but both were resistant to STX.
E. coli harbouring pMAB01 became resistant to KAN,
STR and TOB, but not to STX, suggesting that the
sul1 gene may be non-functional in E. coli.
Conclusions: Genetic exchange, especially the
acquisition of drug-resistance promiscuous plasmids,
couldresultintheemergenceofspecificmycobacterial
strains with altered resistance profiles, which might
be better adapted to cause human disease.
OP75
MOLECULAR CHARACTERIZATION OF MyCOBaCTERIUM avIUM CLINICAL STRAINS FROM
ST. PETERSBURG, RUSSIA
Daria Starkova1, Tatiana Otten2, Igor Mokrousov1,
Anna Vyazovaya1, Boris Vishnevskiy2, Olga
Narvskaya1
1
Russia
2
Research Institute of phthisiopulmonology, St.
petersburg, Russia
Background: Mycobacterium avium is a typical
environmental
nontuberculosis
microorganism
occasionally causing mycobacteriosis, an infection
of wild and domestic animals, birds, and humans. In
recent 10 years, a steady increase in a number of
laboratory confirmed new cases of mycobacteriosis
particularly in HIv-positive patients has been observed
in Saint Petersburg, Russia.
Goal. Molecular identification and the first insight
to genetic diversity of M. avium strains isolated
from 107 patients (including 32 HIv-positive)
with mycobacteriosis in Saint-Petersburg (20082011).
Design/Methods: A total of 107 human mycobacteria
isolates were cultured from individual patients.
Molecular identification of M. avium isolates was
based on the BstEII and HaeIII PCR-RFLP analysis
of hsp65 gene. The PCR-based detection of IS901
and IS900 was used to distinguish among M. avium
subsp. hominissuis (MAH), avium, paratuberculosis,
and silvaticum strains. Reference strains ATCC
35712 and M. avium paratuberculosis were included
as positive controls. The genetic diversity of M. avium
isolates was studied by vNTR-typing based on 8
vNTR loci: 292, X3, 25, 47, 3, 7, 10 and 32 (Thibault
et al., 2007) and additional 12 MATR loci (Inagaki et
al., 2009). The Hunter-Gaston Discrimination Index
(HGDI) was used to assess the discriminatory power
of vNTR-typing. A Neighbour Joining (NJ) tree based
on 20 loci MATR-VNTR profiles was generated by
using algorhythm http://www.miru-vntrplus.org.
Results: The assignment of 107 human
55
mycobacteria isolates to M. avium based on routine
biochemical tests was confirmed by RFLP analysis
of the hsp65 gene. All M. avium isolates were
IS901- and IS900- negative and thus they were
assigned to MAH. The vNTR-typing based on 8- and
12-polymorphic loci allowed for the detection of 39
patterns among 107 MAH isolates. The 27 isolates
had unique vNTR patterns and 80 isolates grouped
into 12 clusters. The three major genotypes were:
22221128223134532443, 24221128213134532443,
and 2533112’8423132432222 including 44 (41.1%),
8 (7.5%), and 7 (6.5%) isolates, respectively. The
7 strains of the cluster 2533112’8423132432222
possessed a truncated locus TR10 (allele 2´) due to
the15bpdeletioninthefirstoftwotandemrepeats
(GenBank accession no. Jq918769.1). Nine clusters
included 2-4 isolates. The highest HGDI values for
the particular loci were as follows: 0.61 (TRX3), 0.53
(MATR-11), 0.362 (TR25, MATR-4), 0.35 (MATR-14
and MATR15); loci 3, 7 and 13 were monomorphic.
The NJ tree (PAuP* package) based on 20 loci
showed that recently evolved vNTR-types were
predominant in the MAH population studied and no
association between M. avium vNTR type and HIv
status of patient was observed.
Conclusions: Taking into account the diversity of
HGDI values for the particular vNTR loci the improved
vNTR-typing scheme of Russian MAH isolates of
different origin is underway.
OP113
WHOLE GENOME SEqUENCING PROVIDES EVIDENCE FOR TRANSMISSION OF MyCOBaCTERIUM aBSCESSUS BETWEEN CYSTIC FIBROSIS
PATIENTS
Josephine M. Bryant1, Dorothy M. Grogono2,3,4,,
Daniel Greaves4, Juliet Foweraker3,5, Iain
Roddick6, Thomas Inns7, Mark Reacher6, Charles
S. Haworth3, Martin D. Curran5, Simon R. Harris1,
Sharon J. Peacock4, Julian Parkhill1, R. Andres
Floto2,3,4.
1
Wellcome Trust Sanger Institute, hinxton, UK
2
Cambridge Institute for Medical Research,
University of Cambridge, UK
3
Cambridge Centre for Lung Infection, papworth
hospital, Cambridge, UK
4
Department of Medicine, University of
Cambridge, Cambridge, UK
5
health protection agency, addenbrooke’s
hospital, Cambridge, UK
5
hpa health protection agency East of England
Regional Epidemiology Unit, UK
7
hpa, norfolk, Suffolk & Cambridgeshire health
protection Unit, UK
Mycobacterium
abscessus,
an
opportunistic
pathogen, is increasingly being isolated from cystic
fibrosispatientsworldwide.Thismulti-drugresistant
organism is particularly difficult to treat leading to
56
chronic infection and an accelerated decline in
lung function. Prior evidence suggests acquisition
is primarily from the environment with an absence
of patient-patient transmission. Whole genome
sequencing was carried out on 168 samples collected
from 31 CF patients attending Papworth hospital
overa4-yearperiod.Thephylogenyidentifiedthree
sub-species of M. abcessus present in the patient
group. Within each subgroup, different patients had
acquired highly diverse strains indicating independent
acquisition from the environment. Some clustering
of strains between patients was evident, and two
levels could be discerned; highly-related clusters with
different patient samples identical or near-identical;
and more distantly-related clusters with patient
samples differing by approximately 50-200 single
nucleotide polymorphisms. Epidemiological links
consistent with recent transmission were identified
inthefirstbutnotthesecond.Weconcludethatthis
data provides the first convincing evidence for both
recent transmission within the CF centre, and the
presence of two dominant circulating clones. The
exact mechanism of both transmission mechanisms
is yet to be determined, however the strict infectioncontrol procedures imposed and our epidemiological
analysis suggests that it is indirect.
OP159
EMERGENCE OF CLINICALLY RELEVANT NONTUBERCULOUS MYCOBACTERIAL INFECTIONS
IN SAUDI ARABIA
Bright Varghese1, Ziad Memish2, Naela Abouljadayel2, Raafat Al-Hakeem2, Fahad Alrabiah3,
Sahal Al-Hajoj Al-Nakhli1
1
Mycobacteriology Research Section,
Department of Infection and Immunity, King
Faisal Specialist hospital and Research Centre,
Riyadh, Saudi arabia
2
Ministry of health, Riyadh, Saudi arabia
3
Department of Medicine, King Faisal Specialist
hospital and Research Centre, Riyadh, Saudi
arabia
Non-Tuberculous Mycobacteria (NTM) are emerging
around the world due to the higher prevalence of
immunosuppressive illness and therapy. Saudi
Arabia is not an exception as there have been novel
mycobacterial species also identified recently. In
addition the availability of several case reports from
different parts of the country suggested the growing
pathogenicpotentialofNTM.Asthefirstnationwide
study we sought to gain an insight into the species
diversity of NTM clinical isolates. During 2009- 2010,
95 clinical isolates were collected from tuberculosis
reference laboratories of major provinces of Saudi
Arabia and subjected to standardized line probe
assay techniques to identify the species. Diagnostic
guidelines of the American Thoracic Society were
applied to determine the clinical relevance of
respiratory isolates. Species diversity (13 species)
was very high in the study and majorly (61.0%)
consisted of rapid growing NTM’s. The major species
obtained were Mycobacterium abscessus, M.
fortuitum, M.intracellulare followed by M. kansassi,
M. gordanae and M. avium. Of the total, 67.1% were
clinically relevant respiratory infections, 23.2% were
non-respiratory cases and 9.7% were respiratory
colonizers respectively. Coexisting illness was
reported in 53.7% of the studied cases. The major risk
factors observed among the patients were previous
history of tuberculosis, chronic obstructive pulmonary
disorderandhumanimmunodeficiencyvirusinfection.
Thehighrateofclinicallyconfirmedrespiratorycases
showed that the NTM infections are indeed a new
challenge to health authorities. Interesting enough,
thecurrentfindingsareshowinganoppositepicture
of the Western world where M. avium complex and
slow growing NTM’s being the most predominant
non tuberculous mycobacterial respiratory pathogen.
The complexity of species demands an immediate
strengthening of current diagnostic facilities to a more
advanced level in the country.
IGRA AND LATENT TB
OP185
DORMANCY AND RESUSCITATION IN MyCOBaCTERIUM TUBERCULOSIS
Barbara Tizzano1, Matthias Wilmanns2, Stefan
Niemann1
1
Research Institute Borstel, Department of Molecular Mycobacteriology, Borstel, germany
2
European Molecular Biology Laboratory (EMBL)
hamburg, notkestrasse 85, hamburg, germany
Mycobacterium tuberculosis has the ability to survive
in humans for decades without causing clinical
symptoms. The “control” of Mtb infection involves the
formation of granulomatous structures where bacilli
remain viable but in a “dormant” state under limiting
nutrient and oxygen conditions. Although survival
of the bacterium in a dormant state is crucial for tb
disease and transmission to another host, little is
known about the pathobiological mechanism involved.
Previous reports point at proteinaceous factors
denoted as Resuscitation-promoting factors (Rpfs) as
reactivation triggers. However, no systematic study is
available on the activity of these proteins on different
clinical isolates. Our work employed a panel of clinical
isolates belonging to different phylogenetic branches
to set up the Wayne model for hypoxia-induced
dormancy. The aim of our studies was to provide a
wider overview of the non-replicating persistence
stateandtoenlightenlineage-specificdifferencesin
the dormancy shiftdown process. Further goal of our
workwastocorrelatedifferentstrainstostrain-specific
reactivation responses and to identify transcriptional
changes occurring in early stages of resuscitation.
Asafirststep,wesetupamodifieddormancymodel
optimized for medium throughput investigation of
clinical isolates. After testing several media and
growth conditions, dormancy experiments have been
successfully carried out with laboratory strain H37Rv
and a clinical isolate of the Haarlem lineage. Both
strains showed the typical growth curve (obtained by
OD and confirmed by c.f.u.) with Non-ReplicatingPersistence stages NRP1 and NRP2. Subsequently,
the effect of resuscitation promoting factor RpfB was
tested by treating “dormant” cultures with different
concentrations of the protein. Both strains showed a
faster resuscitation in response to RpfB. In order to
perform transcriptome analysis on bacteria during the
shifdown in the dormant state, RNA was isolated over
different time points. More clinical strains are currently
under investigation.
In conclusion, we have set up a dormancy model
that allows in depth investigation of pathobiological
mechanism involved in dormancy and resuscitation.
First results indicate that clinical isolates are able
to survive the anaerobic conditions leading to the
dormant state and they recover their ability to grow
and divide after treatment with RpfB. Transcriptome
57
analysis is expected to provide a comprehensive in
depth view on gene regulation networks involved in
the resuscitation process in clinical MTBC isolates.
OP234
IMMUNOLOGICAL FEATURES IN CHILDREN
WITH TUBERCULOSIS
Anna Starshinova, Natalia Korneva, Julia
Ovchinnikova, Olga Yakunova, Elena Potapenko,
Irina Dovgaluk
petersburg, Russia
Objectives: to reveal features of immunological
parameters in children with tuberculosis
Materials and methods: 213 patients 3 – 14
years old were examined during 2010-2012 in the
children’s department of Saint-Petersburg Institute
for Phthisiopulmonology. TB diagnosis was decided
based on clinical and X-ray symptoms. After computer
tomography examination (CT) patients were divided
in two groups: I (n=70) – MBT infected without TB
manifestation (control group); II (n=143) – with TB of
intrathoracic lymph nodes. Immunological examination
included: quantiFERON®-TB Gold IT (qFT-G),
Diaskintest (recombinant tuberculosis allergen based
on M. tuberculosisspecificproteins:ESAT-6andCFP10) (DST) – immunological skin-test, tuberculin skin
test (TT), assessment of leucocytes’ subsets (CD3+,
CD4+, CD8+, CD4 +/CD3+, CD8+, CD16+, CD20+,
CD25+, CD95+,HLAII),cytokines-induced(TNF-ά,IL2,IL-4,INF-γ),tbcspecificIgA,IgG,IgM(Anda-tb).
Results: TT in the I group was: <10mm -15.7%, 1015mm- 64.3% (р=0,05, χ2=4.61), 15mm> 20.0%; in
the II group was: <10mm -9.1%, 10-15mm- 48.9%,
15mm>42.0%(р=0,01,χ2=9.96). Rate of qFT-G test
positive results was significantly higher for children
withTB(II)(р=0,01)(76.9%ofcases(II)vs.30.0%of
cases(I));DSTpositiveresultswassignificantlyhigher
forchildrenwithTB(II)aswell(р=0.001)(84.4%of
cases (II) vs. 41.5% of cases (I). Level of CD16+ in the II
group(58.5%(II)vs.2.4%(I),р<0,001, χ2=38,74)was
significantlyhighercomparedtothecontrolgroup(I).
Itwasnotidentifiedstatisticallysignificantdifference
inthetitersofspecificantibodiesbetweengroups.In
both groups the levels of cytokines-induced IL-2 (I
-238,90±24,3; II -223,05±30,33), IL-4 (I - 1,61±0,12;
II-1,75±0,19)andIFN-γ(I-20454,13±1314,57;II-
19082,95±1723,69) were higher normal ranges.
Conclusions: children with tuberculosis are
characterized by higher level of TT, positive qFT-G,
Diaskintest, higher level of CD16+ which may
be considered thereby as the most informative
immunological tests for TB disease diagnosis in
children.
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ABSTRACTS OF POSTER PRESENTATIONS (P)
M. TUBERCULOSIS GENOMICS
P24
P42
FITNESS OF MyCOBaCTERIUM TUBERCULOSIS
WITH MUTATIONS IN THE RPSL, RRS, GIDB AND
RPOB GENES
DECIPHERING THE EVOLUTIONARY HISTORY
OF THE M. TUBERCULOSIS LATIN AMERICANMEDITERRANEAN (LAM) GENOTYPE
Fernanda Spies1, Andrea Von Groll1, Andrezza
Ribeiro2, Daniela Ramos1, Marta Ribeiro3, Anandi
Martin4, Juan Carlos Palomino4, Maria Lucia
Rossetti2, Pedro Almeida da Silva1, Arnaldo Zaha2
1
Universidade Federal Do Rio grande, Rio
grande, Brasil
2
Universidade Federal Do Rio grande Do Sul,
porto allegre, Brasil
3
Fundação Estadual para produção e pesquisa
em Saúde, porto allegre, Brasil
4
University of gent, gent, Belgium
Anzaan Dippenaar, Rob Warren, Nico Gey van
Pittius
Stellenbosch University, Faculty of health
Sciences Stellenbosch University, Tygerberg,
South africa
Background: It is often assumed that drugresistant microorganisms pay a physiological cost
for the acquisition of drug resistance. In this study
to investigate fitness and drug resistance, we
selected 78 M. tuberculosis strains obtained from
southern Brazilian patients during the period 20052006. The strains were from different lineages, and
have different resistance profiles and mutations in
the rpsL, rrs, gidB and rpoB genes. We evaluated
two parameters, namely the growth curves and the
growth in competition.
Results: Strains with mutation K43R in rpsL had no
difference in growth compared to the susceptible
strains with no mutations and the strains with mutation
S531L in rpoB grew even faster than the susceptible
strains in the logarithmic phase of growth. In the
competitions assays, between the studied strains,
the susceptible strains always grew better than the
drug-resistant strains. We also studied the different
lineages, and according to the growth curves the
LAM strains grew slower than the non-LAM in the
lag phase but the difference in the growth index was
not statistically significant. During the competitions
the non-LAM strains produced more CFus than
the LAM strains, suggesting that LAM strains could
be more frequent due to a putative adaptation to
Brazilian population rather than this genotype confer
an advantage for growth.
Conclusion: Our results suggest that heterogeneity in
fitnessisinfunctionofthedrug-resistancemutations
and the strain genetic background.
Background: Mycobacterium tuberculosis strains
which have evolved over the past 40 000 years
can be grouped into different lineages, sublineages
and strains by standardised genotyping methods.
The Latin American-Mediterranean (LAM) genotype
causes approximately 15 % of tuberculosis (TB) cases
worldwide and is the major cause of TB in Southern
Africa and South America. The frequency at which
different strains occur in these regions is thought to
reflect a combination of host-pathogen compatibility
and virulence.
Hypothesis: The pathophysiological characteristics
of members of the LAM genotype correspond to
changes in the proteome as a result of altered gene
expression derived from genomic polymorphisms.
Aim: To identify genetic differences in a selection of
M. tuberculosis strains that will provide a platform
for the elucidation of phenotypic variation among
members of the LAM genotype.
Methods: DNA from M. tuberculosis clinical isolates
were subjected to whole genome sequencing on
the Illumina HiSeq2000 platform, using a pairedend approach, with 500 base fragment sizes which
results in insert sizes between 350 and 550 bases.
Whole genome sequences from 11 M. tuberculosis
strains representing six different sublineages of
the LAM genotype were analysed and 33 non-LAM
strains were compared to the reference strain H37Rv.
A combination of open source software packages
was used with optimised parameters, together with
in-house developed scripts to compile a customised
pipeline for the analysis of mycobacterial genomes,
with specific focus on variant calling. The depth of
coverage for all the isolates was above 100. This,
together with high quality sequencing data, ensured a
highlevelofconfidenceforidentifyingvariationinthe
genomes of interest. Phylogenetic reconstructions
were done using open source model selection- and
computational phylogeny software packages.
Results: Thiscomparativegenomicsstudyidentified
59
a total number of 10 675 SNPs in 44 strains of M.
tuberculosis analysed. Phylogenetic reconstruction
based on these SNPs were congruent with the
previously described principle genetic groups 1,2 and
3,whilethephylogenyoftheLAMgenotypeidentified
showedthepresenceofthe6sublineagesasdefined
by IS6110 RFLP analysis.
Conclusion:
This is the first comprehensive reconstruction of
phylogenetic relationships focussing on the LAM
genotype of M. tuberculosis. This study provides the
basis for understanding the genotype and phenotype
of the LAM genotype of M. tuberculosis Novel insight
into the phylogenetic history and the biology of
virulence and pathogenicity of this group will provide
opportunity for the identification of new drug and
vaccine targets as well as providing potential novel
genetic markers for this prevalent group of strains.
P44
CHARACTERIZATION OF ExTENSIVELY DRUGRESISTANT MyCOBaCTERIUM TUBERCULOSIS
IN SIBERIA
Maya Dymova1, Olga Alkhovik2, Andrey
Cherednichenko2, Tatyana Petrenko2, Maxim
Filipenko1
1
Siberian Branch Russian academy of Science,
Institute of Chemical Biology and Fundamental
Medicine, novosibirsk, Russia
2
The Ministry of health and Social Development,
novosibirsk Tb Research Institute, novosibirsk,
Russia
The emergence of extensively drug-resistant
TB (XDR-TB) is widely considered a serious
threat to global TB control. Although molecular
characterization of drug resistance-associated
mutations in multidrug-resistant isolates in Siberia
has been made, mutations in XDR isolates and their
genotypes have not been reported previously. In this
study,weidentifiedandcharacterized29extensively
drug resistance Mycobacterium tuberculosis isolates
from clinical isolates in Siberia. The most prevalent
mutationsinvolvedinisoniazid,rifampicin,ofloxacin,
streptomycin resistance were Ser315Thr in katG
gene, Ser531Leu in rpoB gene, Asp94Gly in gyrA
gene, Lys43Arg in rpsL gene, Glu92Asp in gidB
gene respectively. vNTR – typing and deligotyping
revealed that 93% belonged to Beijing family, 11 of
them belonged to highly virulent type M11 (39.2 %).
We also found two isolates belonged to LAM family,
the second most common family in Russia, as well
as Beijing-associated to multi drug resistance. Due to
theepidemiologicalsignificanceofbothfamiliesitcan
be recommended deligotyping as a method of use
for prescreening, and then vNTR-typing, for a more
detailed differentiation of isolates M. tuberculosis.
Our result highlights the need to reinforce the TB
60
policy in Siberia with regard to control and detection
strategies.
P90
LABEL-FREE AND MULTIPLExED
ELECTROCHEMICAL DETECTION OF
PCR FRAGMENTS ON SCREEN-PRINTED
ELECTRODE ARRAYS. APPLICATION TO
SPOLIGOTYPING OF MyCOBaCTERIUM
TUBERCULOSIS
Viet Hai Le1, Steeve Reisberg1, Michel
Gomgnimbou2, Christophe Sola3, Minh Chau
PHAM1, Benoit Piro1
1
Université paris-Diderot, Sorbonne paris Cité,
paris, France
2
Université paris-Sud-Cnrs, Umr8621, Institut
de génétique et Microbiologie, the Infection,
genetics, Emerging pathogens (Igepe) Team,
Orsay, France
3
et Microbiologie, Infection genetics Emerging
This work presents the development of a DNA
electrochemical biosensor based on label-free and
signal-on hybridization detection. Electrodes were
modifiedbyanelectroactiveultrathinfilmobtainedby
co-electro-grafting of a mixture of diazonium salts
(3-aminothiophenol-5-hydroxy-1,4-naphtoquinoneJug-Ph and 4-aminobenzoic acid-Ph-CooH).
Oligonucleotide capture probes modified on their 5’
end with an amino group were covalently grafted on
the Ph-COOH functions, while the Jug-Ph groups
were used as the redox transducers, which translate
hybridization into changes in current. These changes
were monitored using square wave voltammetry (SWv).
Results showed very good reproducibility, excellent
selectivity (discrimination of a SNP) and sensitivity (10
pM), not only with synthetic oligonucleotides targets,
but also with PCR products. Electrodes have been
implemented in a network of 81 independent screenprinted electrodes for application to a specific case:
theidentificationofcladesofM. tuberculosis via the
direct electrochemical detection of 43 sequences,
amplified by PCR, constituting the CRISPR locus
of this bacterium (spoligotyping method). The array
isimplementedaspartofaflowcellandconnected
to a multiplexing system enabling a potentiostat to
sequentially address each of the electrodes. A series
of electrochemistry experiments which previously
could take several tens of hours is now reduced to
a few minutes. Strains of different lineages were
investigated: M. africanum, Beijing, LAM, S, EAI. It is
shown that current intensity changes are specific to
the target sequence, that is of the lineage and strain
from which is produced the CRISPR-PCR product.
The identification of Mycobacterium tuberculosis
at the sub-lineage level could in some cases be
considered as a surrogate marker of tuberculosis
drug resistance and be important both for patient
treatment choice and outcome as well as for public
health.[1] q.D. Zhang, G. March, v. Noël, B. Piro, S.
Reisberg, L.D. Tran, L.v. Hai, E. Abadia, P.E. Nielsen,
C. Sola, M.C. Pham, Biosensors and Bioelectronics
(2012) 32, 163–168
P94
MOLECULAR IDENTIFICATION OF
MyCOBaCTERIUM TUBERCULOSIS COMPLEx
SUBSPECIES IN PROVINCE OF MODENA, ITALY
Giulia Fregni Serpini1, Sara Tagliazucchi1, Giulia
Forbicini1, Nadia Nanni1, Rita Magnani1, Anna
Fabio1, William Gennari1, Anna Maria Teresa
Sabbatini1, Antonella Grottola2, Monica Pecorari1
1
Microbiology and virology Unit, Modena
University hospital, Modena, Italy
2
University of Modena and Reggio Emilia,
Modena, Italy
The Mycobacterium tuberculosis complex (MTC)
include the subspecies M. tuberculosis, M. africanum,
M. microti, M. bovis, M. bovis BCG, M. caprae, M.
canettii and M. pinnipedii that are characterized by
high similarity (99,9%) at the nucleotide level and
identical 16S rDNA sequences. Nevertheless, each
of the MTC subspecies is known to infect humans
even if they show different pathogenicity and drugs
susceptibility. Therefore, the identification of clinical
MTC isolates at the subspecies level is required in
order to collect informations to enable appropriate
patient treatment and public health measures.
The biochemical tests of MTC don’t allow the
subspecies’sidentification,thereforeitneedstouse
molecular methods. The molecular tests based on
commercial probes used for diagnosis routinely don’t
recognize all subspecies. The PCR-based MTC typing
method using chromosomal Regions-of-Difference
(RD4/Rv1510, RD7/Rv1970, RD1/Rv3877-Rv3878,
RD9/Rv2073c, RD12/Rv3120) and specific genic
loci (16S rRNA, cfp35/Rv0577, IS1561) has been
described by Huard and others. This method allows
theidentificationofMTCsubspeciesonthebasisof
theamplificationpatternsobtained.
The aim of this study is to distinguish the subspecies
in MTC drug-resistant strains isolated in the
laboratory of Microbiology and virology of Modena,
Italy, between 2008 and 2012. For this purpose, we
considered 35 MTC drug resistant strains comprising
25 (71,4%) monoresistant to isoniazid (INH) and 10
(28,6%) multi-drug-resistant (MDR).
Among the INH resistant strains 23 (92%) were
identifiedasM. tuberculosis. Out of these, 20 showed
the presence of all the RDs, while 3 strains showed
each the lack of one target amplicon, specifically
RD7, RD1 and IS1561.
Finally2strains(8%)wereidentifiedas M. africanum,
in particular one of these was found to be M. africanum
subtype Ia ( West African 2) for the absence of the
RD7 and RD9 target amplicons and the other strain
M. africanum subtype Ib ( West African 1) for the
absence of the only RD9. All the MDR strains were
identified as M. tuberculosis. Nine of these showed
all RDs while one was lacking of the RD1 target
amplicon.
The lack of RD7 and IS1561 target amplicons in M.
tuberculosis strains was previously described by other
authors while the absence of RD1 target amplicon
remains to be characterize. Further investigations
will be required to delineate the phylogeny of our
M. tuberculosis strains. In particular: (i) the Single
Nucleotide Polymorphisms analysis in katG and gyrA
genes will allow to characterize the Mycobacterium
tuberculosis strains as belonging into one of three
Principal Genetic Groups (PGG1b, PGG2 and
PGG3); (ii) the study of the region TbD1 (mmpl6
gene) will allow the distinction between strains of M.
tuberculosis Ancestral and Modern inside the PGG1b
group.
P108
UNUSUAL LARGE-SCALE CHROMOSOMAL
REARRANGEMENTS IN MyCOBaCTERIUM
TUBERCULOSIS BEIJING B0/W148 CLUSTER
ISOLATES
Egor Shitikov1, J Bespyatykh1, D Ischenko1, I
Karpova1, E Kostryukova1, Y Isaeva2, E Nosova2,
A Vyazovaya3, I Mokrousov3, O Narvskaya3,
B Vishnevsky4, T Otten4, V Zhuravlev4, P
Yablonsky4, E Ilina1, V Govorun1
1
Research Institute of physical Chemical
Medicine, Moscow, Russia
2
Moscow Scientific-Practical Center of Treatment
of Tuberculosis of Moscow healthcare, Moscow,
Russia
3
Russia
4
Research Institute of phtisiopulmonology, St.
petersburg, Russia
Mycobacterium tuberculosis (MTB) Beijing family
isolates are geographically widespread, often
hypervirulent and associated with drug resistance.
One fourth of Beijing genotype strains circulating
in Russia belongs to the so called B0/W148 clonal
group according to restriction fragment length
polymorphism analysis. The aim of the present study
was to investigate features of these endemic strains
on a genomic level.
Four MTB isolates of the Beijing B0/W148 cluster were
sequenced using a pyrosequencing approach (454/
Roche FLX) with greater than 10-fold of coverage.
All individual reads generated using the 454 platform
were mapped to H37Rv genome using the 454 GS
Reference Mapper (Roche 454 Life Science, uSA).
61
The data obtained was compared with published
genomes of the MTB strains, including the one of
W-148 from the same B0/W148 group. Phylogenetic
tree was built based on overall SNPs extracted from
genomic DNA sequences after excluding SNPs for
PE-PPE and PGRS protein families by using the
MEGA 4 program.
Phylogenetic analysis demonstrated a close similarity
between the genomes of four Beijing B0/W148 strains
under study and published earlier genome of W-148
strain. It’s gave us an opportunity to analyze structural
genomic rearrangements within this group. Whole
genome alignment between W-148 and the reference
H37Rv MTB strain revealed two large chromosomal
inversions in the W-148 genome. According to
our hypothesis the largest inversion reversed the
orientation of 3 megabase (Mb) of the chromosome.
The second one occurred in the previously inverted
region and partly restored the orientation of 2.1 Mb inner
segment of the chromosome. These two inversions
wereflankedbypartialorcompletecopiesofmobile
genetic element IS6110 and touched large parts of
genome. Detailed PCR-analysis of our sequenced
strains (n = 4) revealed the rearrangements in their
genomes identical to those ones found in W-148
strain.
This is another example of genomic rearrangements
in the MTB in addition to the published recently
cases of large-scale duplications. These events
occurred in the similar region of genome, which leads
to the assumption that this region is unstable. The
described cases suggest that large-scale genomic
rearrangements in the currently circulating MTB
isolates may occur more frequently than previously
considered, and we hope that further studies will help
to determine the exact mechanism of their formation.
P123
DETECTION OF MUTATIONS BEYOND THE ‘HOTSPOT’ REGIONS OF FIRST- AND SECOND-LINE
DRUG RESISTANCE GENES IN ExTENSIVELY
DRUG-RESISTANT MyCOBaCTERIUM
TUBERCULOSIS ISOLATES USING WHOLE
GENOME SEqUENCING
Asho Ali1, Zahra Hasan1, Taane Clark2, Ruth
McNerney2, Kim Mallard2, Mridul Nair3, Arnab
Pain3, Rumina Hasan4
1
The aga Khan University, Karachi, pakistan
2
London School of hygiene and Tropical
Medicine, London, UK
3
King abdullah University of Science and
Technology, Thuwal, Saudi arabia
4
Department of pathology/Microbiology, The aga
Khan University, Karachi, pakistan
Molecular detection of drug resistance in
Mycobacterium tuberculosis (MTB) is based on
identificationofcommonmutationsindrugresistance
62
conferring genes. Whole genome sequencing (WGS)
has facilitated the identification of synonymous and
non-synonymous single nucleotide polymorphisms
(SNPs), as well as other variations e.g., deletions in
MTB. Geographical variations in SNPs highlight the
importance of understanding region-wise variations.
We performed WGS of extensively drug-resistant
(XDR) (n=37) and susceptible (n=5) MTB isolates from
Pakistan. Thirty-seven genes conferring resistance to
firstandsecondlinedrugswereanalyzed.
Over 150 SNPs (>60% previously unreported)
were identified, including 79 in genes resulting in
resistance to; rifampicin (rpoB), isoniazid (katG,
fpbC, Rv1592C, ndh, Rv2242, fabD, kasa, accD,
oxyR, fadE24, and nat), streptomycin (rrs (500
region), and gidB),pyrazimamide (pnca), ethambutol
(emba, embB, embC, embR, Rv3124, rmlD, inia,
iniB, iniC, and manB) and 22 SNPs associated with
genes conferring resistance to second line drugs;
ofloxacin(gyrA and gyrB), aminoglycosides (tlyA and
ethionamide, ethA and fabG1).
Not all of the above gene targets are included
in commercially available molecular diagnostic
assays for detection of drug resistance in MTB
(i.e., GeneXpert MTB/RIF assay, Haines line probe
assays; MTBDRplus and MTBDRsl). Based on our
data of these XDR-TB strains we observe that the
commercial assays would have missed isoniazid
resistance in 11%, fluoroquinolone resistance in 22
% and aminoglycoside resistance in 19% of MTB
isolates. Therefore, inclusion of additional SNPs
for drug resistance conferring genes are required
to improve molecular methods for MTB resistance
detection.
P248
COMPARISON OF TWO COMMERCIAL
MOLECULAR ASSAYS FOR THE DETECTION OF
TUBERCLE BACILLI IN PARAFFIN-EMBEDDED
TISSUES
Nataša Fajfar, Manca Zolnir Dovc
1
University Clinic of Respiratory and allergic
Diseases, golnik, Slovenia
In terms of histopathologic diagnosis, tuberculosis (TB)
can be diagnosed only as “a chronic granulomatous
inflammation,suggestiveofTB”onaroutinesurgical
pathology report. However, histopathologic features
ofchronicgranulomatousinflammationcanbefound
in various conditions and diseases other than TB.
Therefore other tests should be performed to enable
a definitive diagnosis of TB. Molecular techniques
for the detection of mycobacteria in histopathological
specimens have been introduced as a powerful
supplement to conventional histopathological
diagnosis allready a while ago.
The aim of our study was to evaluate the performance
of Cepheid GeneXpert MTB/RIF assay (GX) for the
detection of M. tuberculosis complex (MTBC) in
paraffin-embedded tissue samples, without special
treatment of tissue to extract DNA, in comparison to
the Gen-Probe Amplified M. tuberculosis Direct test
(MTD), using culture combined with final diagnosis as
a reference method.
A total of 58 formalin-fixed, paraffin-embedded tissue
samples obtained from 45 patients were included in
our study. Deparaffinized slices of tissue were obtained
on glass from Laboratory for Pathology at our clinic.
Tissue blocks ranged in age from 1 to 8 years. Out
of 58 samples, 23 were considered as TB positive
cases and 35 as TB negative cases. Fine powder
of scraped tissue was collected and stored in PBS
buffer for each sample. Tissues in PBS were tested
with MTD and GX assays. These were performed
according to the manufacturer’s instructions.
Out of 58 tissue samples 11 were positive and 30
negative by GX and MTD. Sixteen samples were
positive by MTD only, and 1 by GX only. From 36 TB
positive cases MTD detected MTBC in 27 samples
(75%) but GX in only 12 (33.3%). In comparison
to culture combined with clinical diagnosis, the
sensitivity, specificity, positive predictive value and
negative predictive value were 80%, 100%, 100%
and 71% for the MTD and 60%, 100%, 100% and
47.8% for the GX, respectively.
The results of our study show that commercial
molecular assays can be used as additional means
to confirm the diagnosis of TB from histopathological
specimens. MTBC infection can be detected with a
much higher analytical sensitivity with the MTD than
the GX test. It can therefore be concluded that with
highly sensitive MTD assay, the diagnostic accuracy
can be increased. However, the sensitivity of GX
assay performed from histopathological specimens is
still not sufficient for a final decision of a suspected
TB. Further studies are needed to evaluate the
performance of the GX test for the detection of MTBC
in paraffin-embedded tissues with pretreatment of
tissue samples to extract DNA.
63
P5
PREVALENCE OF HAARLEM FAMILY IN
MyCOBaCTERIUM TUBERCULOSIS IN WORLD
POPULATION: SYSTEMATIC REVIEW AND
META-ANALYSIS
Rashid Ramazanzadeh, Daem Roshani
Cellular & Molecular Research Center and
Microbiology Department, Faculty of Medicine,
Kurdistan University of Medical Science,
Sanandaj, Iran
Background: One of the most prevalent genotype
of Mycobacterium tuberculosis in global population
is Haarlem family that are associated with drug
resistance The aim of this study was to determine
the prevalence and distribution of Haarlem family in
the world using meta-analysis based on systematic
review of articles published.
Methods: All original articles published in literature
database including PubMed, Science direct, Web of
Science, Google Scholar, Biological abs, Iranmedex,
and SID systematically reviewed prevalence of
Beijing family. Data analyzed using meta-analysis
with random effects models.
Results: Final analyses included 78 samples that
have been selected from 2162 studies. Overall
Haarlem family prevalence in world was estimated to
be 8.6% (95% CI 8.3–8.9). Corresponding estimates
by continent were Europe 13.8% (13.1–14.5), Asia
9.5% (9–10.1), America 6.8% (6·2–7.4) and Africa
4.2% (3.5–5).
Conclusions: According to the results, this genotype
is prevalent in European countries. Effective control
program is needed in world to control the spread of
drug resistance strains specially Haarlem family.
Keywords: Haarlem, M. tuberculosis, Prevalence,
Meta-analysis
P26
GENOTYPING OF MyCOBaCTERIUM
BOvIS FROM FARMED ELK IN KOREA BY
SPOLIGOTYPING AND VARIABLE NUMBER
TANDEM REPEAT ANALYSIS
Jae Myung Kim1, Yun-Ho Jang1, Yoonra Jang1,
Soyoon Ryoo1, Narae Kim1, Jae Min Jang1, ShinSeok Kang2, Hyun Sub Byun2, Suk Chan Jung1
1
animal, plant and Fisheries Quarantine and
Inspection agency, anyang, South Korea
64
Chungbuk Institute and veterinary Research,
Changwon, South Korea
2
Bovine tuberculosis (BTB) caused by Mycobacterium
bovis (M. bovis) is primarily a disease of ruminants,
particularly cattle, and is endemic in South Korea.
The aim of this study was to identify and genetically
characterize the M. bovis isolates circulating in farmed
deer in South Korea. Genotyping of the M.bovis
isolates from farmed deer with visible TB lesions was
carried out using two molecular genetic techniques,
spoligotyping and mycobacterial interspersed
repetitive uints-variable number of tandem repeat
(MIRu-vNTR). There are only two spoligotyping
patterns that predominate in farmed deer; strains with
spoligotype pattern SB0140 was the most common
(60% of strains) followed by SB1040 with 8(40.0%)
isolates. These clusters may reflect common
historical origins of M. bovis pattern in South Korea.
Genetic characterization of 21 M. bovis isolates via
the 14 MIRu-vNTR typing distinguished 10 patterns.
The most discriminatory locus for the M. bovis
isolates in Korea was quB 3336 (h=0.57). MIRu 31,
quB 47, vNTR 2401 and 3171 also showed high
discriminatory power (h=0.47). Phylogenic analysis
of M. bovis isolates based on spoligotyping and
MIRu-vNTR separated the isolates into 9 genotypes.
Significant diversity among the M. bovis isolates
circulatinginfarmedelkwasidentifiedwithSB014042532755434343 genotype being the most frequent
strains. In conclusion, spoligotyping and MIRu-vNTR
of M. bovis shows great potential as a molecular
typing tool for characterizing the epidemiology of M.
bovis animal infections in Korea.
P48
MOLECULAR CHARACTERISTICS OF
MyCOBaCTERIUM TUBERCULOSIS BEIJING
GENOTYPE ISOLATES FROM RUSSIA
Anna Vyazovaya1, Igor Mokrousov1, Natalia
Solovieva2, Tatiana Otten2, Boris Vishnevskiy2,
Olga Narvskaya1
1
Russia
2
petersburg, Russia
Background: In Russia, Mycobacterium tuberculosis
strains of the Beijing genotype constitute about half
of the pathogen’s population. They are associated
with multiple drug resistant (MDR) tuberculosis and
increased transmissibility.
Methods: The 105 M. tuberculosis DNA samples
of the Beijing genotype (defined by spoligotyping)
isolates (84 MDR) from epidemiologically unlinked
Russian patients with tuberculosis (2010-2012)
were subjected to IS6110-RFLP and 24-loci vNTRtyping. Data were compared to MIRu-vNTRplus
international database; Hunter Gaston Index (HGI) of
allelic diversity was calculated.
Results: The IS6110-RFLP typing detected 36
different banding patterns (HGI=0.85) containing 1221 bands. Twenty-five isolates had unique patterns
(24%) and 80 strains were grouped into 11 clusters.
The largest RFLP cluster designated B0 included 33
isolateswith17-bandprofilewhichpossessedspecific
upper 2 bands of about 9.2 and 7.1 kb and it was
visually identical to that of internationally recognized
W148 strain. Similar upper double bands were found
in 16-, 17-, 18-, or 19- bands IS6110-RFLP patterns of
16 isolates. The whole set of 49 strains was assigned
to previously described Russian “successful” clone
B0/W148-cluster. The 24-vNTR typing differentiated
105isolatesinto30profiles(HGI=0.82)groupedinto
6 clusters; the largest ones included 21 isolates (57%
MDR) and 38 isolates (92% MDR) that corresponded
to types 94-32 and 100-32, respectively (MIRuvNTRplus). The cluster 100-32 included 78% of the
B0/W148-cluster isolates (68% of them with B0/W148
IS6110-RFLPprofile)whichthuswerecharacterized
by 7 repeats in loci MIRu26 and quB26. Another
vNTR cluster 1066-32 included 4 B0/W148-cluster
isolates characterized by 6 repeats in quB26. The
remaining 7 B0/W148-cluster isolates had unique
MIRu-vNTR pattern, however, they had 7 repeats
in MIRu26 and quB26. The highest HGI for Beijing
strains was achieved for loci quB26 (0.66) and
MIRu26 (0.51) while 12 loci (MIRu2, 4, 16, 20, 23,
24, 27, 39; ETR-B, ETR-C, quB4156, and Mtub30)
showed no variation.
Conclusions: The Russian Beijing strains of the
IS6110-RFLP clonal cluster B0/W148 were divergent
due to polymorphism of MIRu-vNTR loci. However,
most of them (78%) had 7 repeats in MIRu26 and
quB26 and belonged to the internationally recognized
100-32 vNTR cluster.
P61
MyCOBaCTERIUM TUBERCULOSIS INFECTION
IN CATTLE IN CROATIA - CASE REPORTS
Silvio Spicic1, Vera Katalinic-Jankovic2, Ivana
Racic1, Maja Zdelar-Tuk1, Sanja Duvnjak1, Zeljko
Cvetnic1
1
Croatian veterinary Institute zagreb, zagreb,
Croatia
2
Croatian national Institute of public health,
zagreb, Croatia
Among the diseases that have threatened the health
and the lives of people and animals in the past century,
tuberculosis(TB)playedasignificantrole.InCroatia,
incidence of human TB was 15/100.000 in 2011.
Bovine TB occurrence has decreased as a result of a
plannedfightagainstthediseaseinthepastdecades.
Despite the effort, it has not been eradicated yet. In
Croatia, the control of bovine tuberculosis is based
on annual testing of all bovine older than six weeks
by tuberculin skin test. Bovine positive on tuberculin
skin test are slauthered and their organs taken for
bacteriological testing on TB. Methods for molecular
epidemiology are also used in order to enhance the
control of TB.
Described here are the only two cases of M.
tuberculosis infection found in cattle in Croatia. In
2008, one heifer on a small cattle farm was slaughtered
due to positive reaction on bovine tuberculin skin test.
No gross pathological changes were visible on lymph
node and tissue specimens inspected at slaughter.
M. tuberculosis was isolated from bronchial lymph
nodes. Epidemiological investigation based on
MIRU-VNTR typing database enbled us to find the
man – the source of the infection and connect him to
the infection in heifer.
In the second case, in 2010 we found 3 cows positive
onbovinetuberculininthesameflock.Onlychanges
detected at slaughter were enormously increased
bronchial and mediastinal lymph nodes without
visible tubercles. M. tuberculosis was isolated from
bronchial lymph nodes in one cow. Despite extensive
epidemiological investigations and molecular MIRuvNTR genotyping of the cattle strain we were not
able to find identical strain in human database in
Croatia or possible pathways for introduction of
the infection to the heard. This second case of M.
tuberculosis infection in cattle with unknown strain
opens up questions about the sources of infection,
ways of dissemination, but also points to the need
to be more active in case finding of tuberculosis in
humans. Although rare, M. tuberculosis infection
may occur in cattle and other animal species. M.
tuberculosis does not appear to have an indigenous
animal host or reservoir and the animals that become
infected represent most probably accidental hosts.
Humans suffering from active TB are strongly believed
to represent the main source of M. tuberculosis
infection in animals, including cattle. Improvement
of diagnosis of tuberculosis in humans and rapid
detection of carriers ensures limiting of spreading of
M. tuberculosis in humans and in animals.
P67
TRANSMISSION OF M. BOvIS INFECTION
AMONG WILD ANIMALS
Marek Lipiec1, Monika Krajewska2
1
national veterinary Research Institute, pulawy,
poland
2
national veterinary Research Institute,
65
Department of Microbiology, national Reference
Laboratory for Bovine Tuberculosis, pulawy,
poland
Bovine tuberculosis is one of the most import
bacterial diseases among wild animals. Wild boar
TB has already described in Europe, in Bulgaria,
Croatia, France, Germany, Hungary, Italy, Portugal,
Slovakia, Spain and the united Kingdom as the main
wildlife reservoir for tuberculosis. Currently, Poland is
recognizedasofficiallyfreeofthediseaseandbovine
tuberculosis (BTB) cases are rarely found in other
than cattle species. In November 2012, the dead wild
boar (female, ca 4 years old) was found in round of the
the Bieszczady Mounatains of South - East Poland.
In the same area for many years the existence of
bovine tuberculosis in bison (European bison) was
observed. Tissues of the dead wild boar were in
good condition because of low temperatures. The
official veterinarian took for laboratory examination
mediastinal, bronchial and mesenteric lymph nodes
and sent them for testing for tuberculosis. The
samples were subjected to laboratory testing in
National Reference Laboratory for Tuberculosis of
the National veterinary Research Institute (NvRI) in
Pulawy, using the standard procedures. The dissected
tissue material was soaked and homogenized in a 5%
solution of oxalic acid. The resulting supernatant was
removed and the pellet was washed twice with sterile
physiological saline. The rinsed pellet was used for
inoculation of culture media - 3 slants of Stonenbrink’s,
Petragnani’s and Loewenstein – Jensen’s media and
incubated at 37°C. In order to identify species and to
determinethemolecularprofiles,thestrainsobtained
fromthewildboarweretested.Theidentificationand
genotyping of strains was based on spoligotyping
and MIRu-vNTR analysis. The bacterial DNAs were
prepared as reported previously and were submitted
to spoligotyping and 15-loci MIRu-vNTR typing
using published protocols. MIRu-vNTR typing was
performed manually using polymerase chain reaction
amplification of 15 loci. For database comparison,
spoligotypes in binary format were entered in the
SpolDB4 proprietary database of the Pasteur Institute
of Guadeloupe.
It was found that the strain isolated from the wild boar
is identical to the previously isolated strains of bison.
Studies suggest the possibility of transmission of
M.bovis ssp bovis strain from bison to the wild boar.
Thus, despite the elimination of the sick bison herd,
the control of tuberculosis in this area can be very
difficult.
P71
THE USEFULNESS OF ELISA TEST FOR THE
DIAGNOSIS OF BOVINE TUBERCULOSIS IN
POLISH CONDITIONS
66
poland
2
poland
ELISA is now one of the most frequently used
serological tests for the diagnosis of many infectious
diseases of animals. It is now the primary test used in
the diagnosis of cattle paratuberculosis, however, in
the form of a commercial test was not previously used
in the diagnosis of bovine tuberculosis. Diagnostic
of bovine tuberculosis based, in accordance with
international regulations, on the performance of
single or comparative tuberculin test in animals over
6 weeks of age. In case of obtaining a positive or
doubtful result , additional tuberculin test is carried
out after 42 days. During this time, the status of the
herd and trade of animals is suspended. Suspected
animals are eliminated, and the corresponding tissue
samples are analyzed using standard laboratory
methods. The aim of the study was to investigate the
suitability of a commercial ELISA in Polish conditions
for recognition of bovine tuberculosis. Together, in
the two-fold repetitions 50 sera samples from healthy
animals, 44 sera from cows with a positive tuberculin
test, 10 from cattle with anatomo pathological
changes, 15 with positive microbiological test and 16
derived from animals reacting positively in the ELISA
test for paratuberculosis was examined. It was found
that the sensitivity of commercial ELISA reached
90% and was higher than the gamma interferon test
. In the study there were no false positives or false
negatives results. Johne’s disease infection did not
affect the results of the test for bovine tuberculosis.
The specificity of the test is set at 100%.The used
test showed a large utility in the diagnosis of bovine
tuberculosis. This is less time-consuming and laborintensive than the gamma interferon test and the
results are easier to the interpretation. In Polish
conditions can be used as a additional and helper
test in the diagnosis of bovine tuberculosis. Could be
implemented especially after the doubtful results of a
single or comparative tuberculin test.
International law does not allow the use of serological
tests as the only force in the fight against bovine
tuberculosis, but it seems that in the future the ELISA
test may play an important role in clarifying the status
of epizootic bovine herds
1
P76
MIRU-VNTR TYPING REVEALS HIGH
HETEROGENEITY IN A SUPPOSEDLY CLONAL
GROUP OF MyCOBaCTERIUM BOvIS
Sabrina Rodriguez-Campos1, Yurena Navarro2,
Beatriz Romero1, Javier Bezos1, Lucía de Juan3,
Carmen Casal Comendador1, Lucas Domínguez3,
Alexandra Gutiérrez1, Darío García de Viedma4,
Alicia Aranaz3
Centro de vigilancia Sanitaria veterinaria
(visavet), Universidad Complutense de Madrid,
Madrid, Spain
2
Cei Campus Moncloa, Ucm-Upm, Servicio
de Microbiología Clínica y Enfermedades
Infecciosas, hospital general Universitario
gregorio Marañón, Inst. Investigación, Sanitaria
gregorio Marañón, Madrid, Spain
3
Departamento de Sanidad animal, Facultad de
veterinaria, Madrid, Spain
4
Servicio de Microbiología Clínica y
Enfermedades Infecciosas. hospital general
Universitario gregorio Marañón, Madrid, Spain
1
Spoligotyping is often complemented by MIRu-vNTR
typing in order to increase discrimination. In Spain,
SB0121 (SIT481) is the most prevalent spoligotype
(28%) involved in bovine tuberculosis. In this study
we applied MIRu-vNTR typing to a panel of randomly
selected Spanish M. bovis SB0121 isolates to assess
allelic diversity and overall discriminatory power.
Furthermore, MIRu-vNTR was applied to all TBpositive animals from selected farms infected by M.
bovis SB0121 to evaluate the degree of heterogeneity
in supposedly epidemiologically linked isolates.
The samples were obtained between 1997 and 2010
from all over Spain and originated from cattle (n =
244), goat (n = 2), wild boar (n = 7), red deer (n = 2),
fallow deer (n = 2), badger (n = 2) and domestic pig
(n = 2). MIRu-vNTR typing was carried out in a twostep approach: 1) Assessing a random selection of
isolates (n=115) using 9 vNTR loci: ETR-A, ETR-B,
ETR-D, ETR-E, MIRu26, quB11a, quB11b, quB26
and quB3232. 2) Screening of 176 TB-positive
animals from 15 farms for heterogeneity applying the
6 most discriminatory MIRu-vNTR markers; in case
of encountering differences in the genotypes, the
9-loci MIRu-vNTR type was completed to determine
the degree of variation.
Thefirstpanelofisolateswasdividedinto65different
MIRu-vNTR (Mv) types achieving a discriminatory
power D=0.9856. The most discriminatory loci were
in descending order: quB3232, ETR-A, ETR-B,
quB11a, quB26, MIRu26, ETR-D, ETR-E and
quB11b. To determine a more cost-effective typing
format we compared the effect of reducing the
number of markers for the analysis: six-loci approach
D=0.9826, four-loci D=0.9689.
Regarding the second panel screened for
heterogeneity in assumedly homogeneous farms, we
found 5 out of the 15 farms to be truly homogeneous.
In the remaining farms we observed: i) 1 farm with
Mv types differing in only 1 locus, ii) 6 farms with Mv
types differing in 2-6 loci and iii) 3 farms in which Mv
types differing in only 1 locus were found along with
Mv types with variation at more than 1 locus.
The characterisation of the random panel of M.
bovis SB0121 isolates revealed high diversity. This
finding along with the heterogeneity detected in ten
out of the 15 farms under study suggests considering
spoligotyping as limited for source tracking.
Heterogeneity reveals the potential existence of
continuous risk of infection and the involvement of
microevolution events in the analyzed farms.
P77
EVALUATION OF COBAS® TAqMAN MTB FOR
DIRECT DETECTION OF MyCOBaCTERIUM
TUBERCULOSIS COMPLEx IN COMPARISON
WITH THE COBAS® AMPLICOR MTB
Claudia Ritter1, Guido Bloemberg2, Antje Voit2,
Vanessa Deggim2, Erik Böttger1
1
Institut Für Medizinische Mikrobiologie,
Zürich, Switzerland; Nationales Zentrum für
Mykobakterien, zürich, Switzerland
2
Institut Für Medizinische Mikrobiologie, zürich,
Switzerland
The Roche COBAS Amplicor MTB assay, recently
replaced by the Roche COBAS Taqman MTB
assay, was one of the first commercially available
assays for detection of Mycobacterium tuberculosis
complex based on nucleic acid amplification. We
have previously reported on a limited specificity of
COBAS Amplicor MTB assay, in particular for positive
samples with an OD660 < 2.0. using a selected set of
respiratory samples, which were scored false positive
by the COBAS Amplicor test, we here demonstrate
that specificity of the COBAS Taqman assay is
significantly improved. In addition, studying a set
of 133 clinical samples, revealed that the COBAS
TaqmanMTBshowedsignificantlylessPCRinhibition
as compared to the COBAS Amplicor test. An overall
concordance of 98.2 was observed between the
two assays. In a subsequent prospective study we
evaluated the Roche COBAS Amplicor MTB assay
on 1143 clinical specimens, including respiratory
(n=838) and nonrespiratory specimens (n=305).
For respiratory specimens a sensitivity of 89.7%, a
specificity of 100%, a PPV of 100% and a NPV of
99.0% was determined. For nonrespiratory specimens
a sensitivity of 77.8%, a specificity of 100%, a PPV
of 100% and a NPv of 96.8% was determined. We
conclude that the COBAS TaqMan MTB assay is a
significantlyimprovedtoolforthedirectdetectionof
MTB in clinical specimens.
P79
A PUTATIVE COMPENSATORY MUTATION IN
THE RPOC-GENE ExCLUSIVELY DETECTED
IN ISOLATES OF THE RESISTANT EUROPEAN
TUBERCULOSIS (RET) CLUSTER WITH A MDRTB PHENOTYPE
67
Jessica De Beer1, Indra Bergval2, Anja
Schuitema2, Richard Anthony2, Maryse Fauville3,
Beatriz Ferro4, Viviana Ritacco5, Aldert Zomer6,
Jakko van Ingen7, Dick van Soolingen8
1
Environment (Rivm), Bilthoven, netherlands
2
Kit Biomedical Research, Royal Tropical
Institute, amsterdam, netherlands
3
Tuberculosis and Mycobacteria, Scientific
Institute of public health, Brussels, Belgium
4
Tuberculosis area, Cideim, Cali, Colombia
5
Instituto nacional de Enfermedadeinfecciosas
anlis “Carlos g. Malbran”, Buenos aires,
argentina
6
Centre for Molecular and Biomolecular
Informatics, Radboud University nijmegen
Medical Centre, nijmegen, netherlands
7
Department of Medical Microbiology, Radboud
University nijmegen Medical Centre, nijmegen,
netherlands
8
mono-resistant and sensitive isolates with other vNTR
profiles, as well as 62 isolates of other genotypes
from Argentina, Belgium and The Netherlands. The
rpoC F452S mutation was only detected in isolates of
thelargeEuropeanoutbreakclusterandspecifically
those with an MDR phenotype that contain an embB
M306v mutation.
The combination of the most favorable mutations
associated with rifampicin and isoniazid resistance;
rpoB S531L and katG S315T, and the putative
compensatory mutation F452S in the rpoC-gene
likely provides this MDR outbreak strain a fitness
advantage. This “super-spreader” strain, which has the
sameVNTRprofileassuccessfullyspreadingMDRTB clones in former Soviet union states deserves
continued monitoring and fundamental research into
its evolutionary development, as the spread of such
typeofstrainsmaynegativelyinfluencethesuccess
of the global TB control worldwide.
Two consecutive European projects on the molecular
surveillance of MDR-TB from 2003-2007, using first
IS6110 restriction fragment length polymorphism
(RFLP) and later 24-locus variable number of
tandem repeat typing (vNTR), detected the same
large cluster of the Beijing genotype (Eu0051); this
cluster comprised 61% (175/288) in the first and
49.9% (n=470/941) of all clustered MDR-TB isolates
in the second project. Thus this resistant European
tuberculosis (RET) cluster outbreak continued to
spread in the European region and is responsible
for a significant proportion of all clustered MDR-TB
cases. This worrying observation prompted research
on the selective advantages of these strains.
The RET outbreak isolates were characterized by
GenoType MTBDRplus reverse line blot method
(HAIN Lifescience, Nehren, Germany), identical
mutations associated with resistance for rifampicin and
isoniazid in 47 of the 48 isolates tested (rpoB S531L
and katG S315T, respectively). It has been shown in
previous studies that these mutations yield the lowest
fitnesscostcomparedtootherresistance-associated
mutations.Themutationsrelatedtofluoroquinolone,
aminoglycoside and ethambutol resistance showed a
wide variation.
Illumina whole genome sequencing, performed by
Baseclear (Leiden, The Netherlands), of 9 outbreak
isolatesidentifiedaputativecompensatorymutation
in the rpoC gene. This mutation, F452S, was not
found in an isoniazid mono-resistant strain and two
sensitivestrainswiththesameVNTRprofileandthus
was restricted to MDR-TB RET isolates, suggesting
this may be a crucial factor in the success of these
MDR-TB strains. Additionally, we screened for this
rpoC F452S mutation in: 31 other isolates of the RET
cluster (15 MDR-TB, 14 INH mono-resistant and 2
sensitive isolates) from 6 European countries, 21
Beijing genotype MDR/XDR M. tuberculosis, INH-
CURRENT STATUS OF BOVINE TUBERCULOSIS
IN POLAND - 3 YEARS AFTER THE
RECOGNITION OF THE COUNTRY FREE OF THE
DISEASE
68
P87
poland
2
poland
1
Bovine tuberculosis is an infectious disease of
cattlewithastrictlydefinedrulesforthecontroland
eradication. For many years Poland has struggled
with bovine tuberculosis. In 2009, based on European
Commission Decision of 23 April 2009, Poland has been
recognized as officially free of bovine tuberculosis.
This recognition does not mean, of course, complete
elimination of the disease in veterinary practice.
According to data for 2009, which recognized Poland
as a country free from bovine tuberculosis, the study
was performed including 111 samples from cattle
suspected of being recognized as a disease (reactive
tuberculin test). The existence of the disease was
found in 60 animals from 12 farms (12 outbreaks).
In another 2010 years the epidemiological situation
deteriorated slightly: a total of 147 samples were
tested, and the disease was found in 18 different farms.
One third of them were located in the northern part
of the Mazowieckie Province, where for many years
the existence of outbreaks of bovine tuberculosis was
observed. In the next year 2011 were similar to the
epidemiological situation: a total of 180 samples were
tested and the existence of tuberculosis reported in
16 outbreaks and in 2012 it were 15. During the study
period, an additional difficulty in the study was the
change in tuberculin diagnostic preparation. Polish
tuberculin, used for many years, was replaced by the
Czech tuberculin, with similar power but with different
injection volumes. There was also a change in the
frequency of field tests performed. Decision of the
ChiefVeterinaryOfficershallbetestedannually20%
of the cattle in the area so that within 5 years the
entire population was examined. In laboratory test the
negative rate (without isolation of strain) ranged from
40to50%.Thisindicatesasignificantproportionof
false-positive results ascertained tuberculin test.
Taking into account all the data obtained during the
period, it is clear that testing for bovine tuberculosis
in Poland and the adopted system allows to maintain
the achieved status of a bovine tuberculosis-free and
allows for controlled combat and further elimination of
the disease in cattle herds.
P106
TRAINING IN BIOSAFETY AGAINST M.
TUBERCULOSIS: ExPERIENCE IN THE
MYCOBACTERIA REFERENCE CENTER AT THE
UNIVERSITY OF CORDOBA, SPAIN
biocontainment levels, protective equipment and
hazardous waste management.
Our proposed security sheet against M tuberculosis
consists of the following sections: description,
feasibility, risks in the laboratory, health surveillance,
exposure control (containment level, collective and
individual protection), handling and storage.
Amongst the several tasks performed by our staff
listed in manual work procedures are established
biosafety schemes which involve safe practices,
use of protective equipment and action to incidents
/ accidents. There is an outline of the procedure for
hazardous waste management.
The staff is trained regularly establishing a simulation
to carry out the emergency plan in the laboratory.
Conclusions: Staff training is essential to prevent
laboratory-acquired
infections,
incidents
and
accidents. This one must include information on
safety practices that should be followed to avoid or
minimize the risk of inhalation and inoculation and
properly decontaminate and also remove infected
material.
P110
Vaquero-Alvarez Esther, Pablo López-Roldan,
Emilio J Aguilar, Fernando Palomares, Francisco
Torralbo, Manuel Vaquero, Manuel J Casal
University of Cordoba, Cordoba, Spain
A NOVEL MODEL TO CONTROL FOR
PATIENT RISK FACTORS IN MEASURING THE
TRANSMISSIBILITY OF MyCOBaCTERIUM
TUBERCULOSIS STRAINS AND LINEAGES
The Mycobacterium Reference Center is a laboratory
with a large workload that performs diverse activities
like direct sputum smear microscopy, preparation
of specimens for use in an automated nucleic acid
amplificationtestcartridge,processingofspecimens
for inoculation on primary culture media, culture
manipulation for identification, DST or line-probe
assays on cultured isolates.
Objective: To present our experience in providing
information and training our staff to prevent
occupational hazards in working with exposure to M.
tuberculosis.
Methodology: Following a risk assessment is directed
a training plan integrated into the Centre prevention
plan, where the workers, the head of the laboratory
and safety technicians are involved. There are
trainingcoursesinfirstaid,fireprotectionandother
specificaboutbiosafety.Thesecoursesof5hoursare
taught by specialists in occupational safety. A safety
data sheet management of M tuberculosis in the
laboratory must be completed and also hazardous
waste management must be reported. From existing
working procedures in the laboratory, each task is
added in a section of safe practices. Finally, we train
the personnel coping emergencies as well as a drill
for training is carried out.
Results: The course, which is celebrated periodically
and attended by all the laboratory personnel, includes
training in biosafety concepts on biological agents,
classification, transmission, universal measures,
Hanna Nebenzahl-Guimaraes1, Martien
Borgdorff2, Jessica de Beer1, Megan Murray3,
Dick van Soolingen1
1
Rijksinstituut voor volksgezondheid En Milieu
(Rivm), Bilthoven, netherlands
2
Municipal health Service, amsterdam,
netherlands
3
harvard School of public health (hsph), Boston,
USa
Background: Host-related risk factors, such as age,
sex and smear positivity, are still considered the
most important factors influencing transmission of
Mycobacterium tuberculosis (MTB). Recent findings
however suggest that the spread of MTB also depends
on bacteriological factors. So far studies looking at
the association between strains or phylogenetic
lineages of MTB and transmissibility have arrived at
differing and often contradictory conclusions. In the
Netherlands all MTB isolates have been subjected
to DNA fingerprinting since 1993 and all patient
information is available. Here we describe a method
for quantifying and controlling for host-related risk
factors in order to go beyond the conventional use
of cluster size and proportion of cases in a cluster as
proxy measures of transmissibility, thus strengthening
the association found with strains and phylogenetic
lineage.
Methods: using spoligotyping and MIRu-typing we
classified MTB isolates from a nationwide database
69
of isolates from the Netherlands into four major
phylogenetic lineages: Euro-American, Central
Asian Strain (CAS), East-African-Indian (EAI) and
Beijing. Isolates with identical IS6110-RFLP or
VNTR fingerprints were classified as clustered, and
Clustering Rates (the proportion of clustered isolates)
was calculated per phylogenetic lineage. Odds Ratios
of host characteristics for clustering were estimated
using a logistic regression model and used as weights
in calculating each patient’s propensity to transmit
(PPT). The geometric mean of PPT values belonging
to a cluster was taken as the overall measure of a
cluster’s propensity to transmit (CPT). An analysis
of variance (ANOvA) was carried out to check
whether PPT values were comparable between the
four lineages. A “high transmissibility” category was
defined as clusters in the bottom 33rd percentile of
CPT values with Cluster Size above average.
Results: Our dataset consisted of a total of 10,389
strains;6,595classifiedasEuro-American,1,327as
CAS, 1,422 as EAI and 1,045 as Beijing. PPT values
from strains of the Euro-American lineage were on
average higher than those of Beijing, CAS and EAI
strains (0.05 level of significance). Clustering Rates
were 60.7% (95% CI, 59.5-61.9) for Euro-American
strains, 49.2% (95% CI, 46.5-51.9) for CAS strains,
51.1% (95% CI, 48.5-53.7) for EAI strains and
49.4% (95% CI, 46.4-52.4) for Beijing strains. In
comparison, the proportion of clusters belonging to
the “high transmissibility” category was 10.4% (95%
CI, 8.6-12.5), 10.5% (95% CI, 6.5-15.7), 14.5%
(95% CI, 10.1-19.9) and 25.8% (95% CI, 19.3-33.3)
for Euro-American, CAS, EAI and Beijing strains,
respectively.
Conclusion: By correcting for host-related factors
in measuring transmissibility of strains in The
Netherlands, the Beijing phylogenetic lineage is
showntobesignificantlymoretransmissiblethanthe
Euro-American, CAS or EAI lineages.
P125
EPIDEMIOLOGICAL ANALYSES OF
TUBERCULOSIS IN ARCHANGELSK, RUSSIA
AND IMPLEMENTATION OF A RAPID ASSAY
FOR DETECTION OF RESISTANCE IN THIS HIGH
BURDEN SETTING
Platon Eliseev1, Andrey Maryandyshev1, Elena
Nikishova1, Irina Tarasova2, Galina Gorina2, Erja
Chryssanthou3, Lars-Olof Larsson4, Malin Ridell5
1
northen State Medical Universiy, archangelsk,
Russia
2
Regional Clinical antituberculosis Dispensary,
archangelsk, Russia
3
Clinical Microbiology, Karolinska Instirute,
Stockholm, Sweden
4
Respiratory Medicine, Karolinska Institute,
Stockholm, Sweden
5
Microbiology; Biomedicine, The University,
gothenburg, Sweden
70
The aim was twofold: to perform epidemiological
analyses of tuberculosis (TB) in the Archangelsk
region with particular focus on multidrug-resistant
(MDR) TB and to evaluate the molecular method
MTBDRplus assay in this high burden setting.
The number of TB cases was 812 (incidence 65/105)
and among these patients, 151 cases were registered
in the penitentiary system (incidence 1162/105).
Most patients were men, 94% had pulmonary TB
and 22% were relapses. Out of all cases, 341 (42%)
were smear positive and thus contagious. 176
(22%) patients had MDR–TB, among which one had
extensively drug resistant tuberculosis (XDR–TB).
The number of cases being both contagious and
MDR–TB was 128 representing 16% of all TB cases
(incidence 10/105). Among these 128 TB patients 37
were relapses representing 26% of all the relapse
cases. The results of MTBDRplus and Bactec MGIT
analyses corresponded in 99%.
In conclusion it can be stated that many TB patients
had relapsing pulmonary TB in Archangelsk. A
large number had contagious MDR-TB thus being
hazardous in society. Many patients were prisoners
demonstration that the TB situation in the prisons is
particularly severe. The analyses showed furthermore
that MTBDRplus is of major value in this setting
P139
ACCURATE AND EFFICIENT IDENTIFICATION
OF DRUG RESISTANCE AND LOCAL MDR
CLUSTER STRAINS OF MyCOBaCTERIUM
TUBERCULOSIS USING AN ADAPTED SNPBASED MULTIPLEx LPA ASSAY
Sarah Sengstake1, Indra Bergval1, Anja
Schuitema1, Kiki Tuin2, Edgar Abadia3, Jessica
De Beer4, Nino Bablishvili5, Nino Bzekalava5,
Nadia Brankova6, Viktoria Levterova6, Rusudan
Aspindzelashvili5, Christophe Sola7, Stefan
Panaiotov8, Dick van Soolingen9, Richard
Anthony1
1
Kit Biomedical Research, Royal Tropical
Institute, amsterdam, netherlands
2
Mrc-holland, amsterdam, netherlands
3
Laboratorio de genética Molecular, Cmbc,
Instituto venezolano de Investigaciones
Cientificas (Ivic), Caracas, Venezuela
4
Environment (Rivm), Bilthoven, The netherlands
national Tb Reference Laboratory, national
Center for Tb and Lung Diseases, Tbilisi,
georgia
6
Environment, Bilthoven, The Netherlands;
national Centre of Infectious and parasitic
Diseases, Sofia, Bulgaria
5
Université paris-Sud-Cnrs, Umr8621, Institut
de génétique Et Microbiologie, the Infection,
genetics, Emerging pathogens (Igepe) Team,
Orsay, France
8
national Center of Infectious and parasitic
Diseases, Sofia, Bulgaria
9
7
Whole genome sequencing (WGS) is likely to be
increasingly applied for characterization of M.
tuberculosis (MTB) isolates. Multiplex SNP-based
assays such as the multiplex ligation-dependent probe
amplification (MLPA) may be complementary to this
approach until all clinical isolates can be subjected to
whole genome sequencing and subsequent analysis.
Whiletheindicativeidentificationofdrugresistance,in
particular to second line drugs, is of major importance
fordiagnosticpurposes;strainidentificationisrelevant
for monitoring of the TB epidemic.
We have developed a multiplex SNP-based assay,
the MLPA, for the characterization of cultured MTB
isolates. The assay targets mutations conferring
resistancetofirst-lineandsecond-linedrugsand
can assign a MTB strains to the six main lineages
of the MTB complex. We strive to continuously
adapt our assay to make use of newly discovered
SNPs conferring drug resistance and for emerging
phenotypic clades.
Here we report of the design and evaluation of
probes to detect mutations in eis, rplC and atpE
conferring resistance to the anti-TB drugs kanamycin,
linezolid and the recently approved drug TMC207,
respectively. Criteria for the inclusion of drug
resistance associated markers was their occurrence
in clinical isolates in addition to in vitro generated
mutants. If several mutated loci were reported the
three most prevalent ones were selected. We have
further included two markers for the identification of
geographically important MDR-TB clusters. Previous
WGS of clustered TB isolates of the largest MDR
cluster in the WHO European Region, identified a
mutation in rpoC to be uniquely associated to this
cluster (see poster by de Beer et al.). The second
marker identifies spoligotype SIT41 belonging to
the TuR lineage which is associated with multi-drug
resistance in Bulgaria. De novo gene sequencing of a
reference panel of strains revealed a mutation in rpfB
asanaccurateidentifierfortheTURlineage.
Here we show that our assay can accurately identify
the presence or absence of the newly included markers
for drug resistance and geographically interesting
MDR-TB clusters. The bead-based MLPA assay
can analyse up to 50 markers simultaneously in one
sample with time to result being between those of line
probe assays and DST. until large-scale sequencing
becomes widely accessible, multiplex SNP-typing
is being applied in The Netherlands, Bulgaria and
Georgia in the laboratory diagnosis of TB.
P149
GENOTYPIC DIVERSITY OF MyCOBaCTERIUM
TUBERCULOSIS IN CONJUNCTION WITH
DEMOGRAPHIC AND EPIDEMIOLOGIC DATA
UNDERLINES MAJOR DIFFERENCES BETWEEN
FRENCH VERSUS FOREIGN-BORN CASES IN
THE RHôNE-ALPES REGION, FRANCE (20002010)
Catherine Pichat 1, David Couvin2, Gérard Carret1,
Véronique Jacomo3, Anne Carricajo4, Sandrine
Boisset5, Jean-Pierre Flandrois1, Gérard Lina1,
Nalin Rastogi2
1
Laboratoire de Bactériologie, Centre de Biologie
Sud, Chu de Lyon-Sud, pierre-Bénite, Lyon,
France
2
Who Supranational Tb Reference Laboratory,
Tb & Mycobacteria Unit, Institut pasteur de la
guadeloupe, abymes, France
3
Service des Mycobactéries, Laboratoire
Biomnis, Lyon, France
4
Laboratoire de Bactériologie, Chu de St Etienne
nord, Saint Etienne, France
5
Laboratoire de Bactériologie, Chu de grenoble,
grenoble, France
As part of a quality and epidemiological observatory
led by various hospitals and laboratories working
on mycobacteria in the Rhône-Alpes region, we
report here the results on the genotypic diversity of
Mycobacterium tuberculosis complex strains isolated
from 2000 to 2010. This study was conducted on 2257
strains (one isolate/patient) of: M. tuberculosis n=2226;
M. bovis n=95; and M. africanum n=36. All strains were
typed by spoligotyping (n=2257), and a significant
proportion by 15-loci MIRu-vNTRs (n=974 or 43.15%).
Comparison of genotypes with the international
SITvIT2 database (the proprietary version housed
at Institut Pasteur de Guadeloupe contained more
than 110,000 isolates from 160 countries) allowed to
draw conclusions on the genotypic diversity of strains
in relation to demographic and epidemiological data.
Spoligotyping-based genotypic lineages in this study
were: T (n=762, 33.76%); Haarlem (n=514, 22.77%);
LAM (n=413, 18.30%, of which n=53, 2.35% were
LAM10-CAM); BOv (n=95, 4.21%); Beijing (n=84,
3.72%); EAI (n=66, 2.92%); S (n=63, 2.79%); AFRI
(n=36, 1.60%); CAS (n=30, 1.33%); X (n=9, 0.40%);
and the Turkey family (previously classified as
LAM7-TuR ; n=7, 0.31%). The most predominant
patterns corresponded to SIT53/T1 (n=346, 15,3%)
> SIT50/H3 (n=166, 7,35%) > SIT42/LAM9 (n=125,
5.5%) > SIT1/Beijing (n=72, 3,2%) > SIT47/H1
(n=71, 3,1%). Thus, the Euro-American lineage
which includes evolutionary recent strains (T, LAM,
Haarlem, X, S) grouped together 1768 or 78.33% of
the strains. Analysis of drug resistance, the location
of disease (pulmonary or extrapulmonary), sex and
age of patients, and the study of phylogenetic and
statistical data set helped draw important conclusions
71
about risk factors for developing the disease. Thus,
resistant strains including MDR forms were most
commonly associated with the age group 21-40 years
(p <0.001) and extrapulmonary tuberculosis was
more prevalent among women, while the pulmonary
form predominated in men (p <0.001). Lastly, BOv
and CAS lineages were particularly prevalent among
patients with extrapulmonary disease (p <0.001).
The origin was known for 927 patients: 376 (40.6%)
French, and 551 (59.4%) foreign-born. Patients of
French origin were relatively older (mean age 58.42
years) than patients of foreign origin (mean age 42.38
years). Africa alone accounted for 70% of patients of
foreign origin, including 39% from the Maghreb and
30.1% from sub-Saharan Africa. Furthermore, AFRI,
EAI, LAM, Beijing, S, and CAS lineage strains were
mostly found in patients of foreign origin while those
belonging to Haarlem, T and BOv lineages were
more common in patients of French origin. Most
importantly, comparison of spoligotypes from foreignborn patients with the SITVIT2 database confirmed
that most were infected with genotypes prevalent in
their country of origin.
P152
MOLECULAR ANALYSIS OF MyCOBaCTERIUM
BOvIS ISOLATES FROM HUMANS IN ITALY:
COMPARISON WITH THE GENOTYPE
DATABASE OF ANIMAL STRAINS
Maria Pacciarini1, Maria Goria2, Giuseppina
Ferraro2, Silvia Tagliabue1, Ester Mazzola3,
Maria Tullia Simonetti4, Paola Dal Monte5, Piera
Mazzone6, Maria Beatrice Boniotti1
1
IzSLER– Centro di Referenza nazionale per la
tubercolosi bovina da M. Bovis, Brescia, Italy
2
IzSpLv, Torino, Italy
3
Ospedale niguarda azienda Ospedaliera,
Milano, Italy
4
azienda Ospedaliera Careggi, Firenze, Italy
5
azienda Ospedaliero Universitaria S. Orsola,
Bologna, Italy
6
IzSUM, perugia, Italy
Spoligotyping and variable Number Tandem Repeats
(vNTR) analysis of M. bovis and Mycobacterium
caprae strains isolated during the national eradication
programme of bovine tuberculosis (bTB) in Italy,
has been routinely performed since 2000. vNTR
typing includes 5 ETRs and 7 MIRu/vNTR markers
(vNTR2163a, vNTR2163b, vNTR4052, vNTR1895,
vNTR3155, vNTR3232, MIRu26) selected for their
high genotypic discriminatory power.
This has enabled to obtain a national animal database
(ITAN-TB) containing information and genotypes of
4291 M. bovis/M. caprae strains isolated from 2500
TB outbreaks as a supporting tool for epidemiological
tracing of TB.
In this study a collection of 141 M. bovis and 2 M. caprae
72
recovered from human patients in Lombardy, Tuscany,
Piedmont and Emilia-Romagna, from 2000 to 2012,
were characterized by the same genotyping method.
Weidentified45spoligotypes,9ofwhich(involving12
strains) never described in the international website
www.M.bovis.org and 14 (involving 17 strains) not
reported in the animal database. The remaining 22
spoligotypes, representing 114 out of the 143 human
strains, were already present in the ITAN-TB.
The most frequent spoligotypes were SB0120,
SB0134 and SB0841 found respectively in 41%,
7,9% and 5,9% of the human isolates and described
as prevalent also in M. bovis population of animal
origin (53,6%, 8,9% and 6,2%).
Combination of the 22 spoligotypes present in the
ITAN-TB, with vNTR analysis discriminated the
114 strains into 46 different genotypes, 31 of which
unique in humans (differ by at least 2 vNTR markers
compared to animal genotypes) and 15 identical or
almost identical (single locus variation) to genotypes
described in TB positive cattle/buffalo herds localized
mainly in Southern Italy. In particular two genotypes
were widespread both in human patients than in
animal database: SB0134-ETR54534-vNTR 10, 4,
5, 4. 3, 5/6, 5 and SB120-ETR45533-vNTR 10, 4, 4,
4, 3, 6, 5/6 (order of markers as described above),
found respectively in 4 and 6 patients and in 42 and
97 TB outbreaks.
Interestingly most of M. bovis human isolates which
showed a correspondence with animal genotype
database were recovered from Italian-born patients.
P153
CHANGES IN MyCOBaCTERIUM
TUBERCULOSIS POPULATION STRUCTURE
IN THE FRENCH DEPARTMENTS OF THE
AMERICAS: A 17 YEARS OVERVIEW
Julie Millet1, Elisabeth Streit1, Anne Gaël Bomer1,
Franzisca Schuster1, Nalin Rastogi2
1
Institut pasteur de la guadeloupe, abymes,
France
2
Who Supranational Tb Reference Laboratory,
Tb & Mycobacteria Unit, Institut pasteur de la
guadeloupe, abymes, France
In the present work, we analyzed Mycobacterium
tuberculosis population structure in the 3 French
Departments of the Americas over a 17 years
period in conjunction with spatiotemporal patient’s
demographics, bacteriologic data, as well as an
assessment on drug-resistance and multidrugresistance (MDR). A total of 1239 M. tuberculosis
isolates were collected from patients living in
Guadeloupe, Martinique and French Guiana from
January 1995 to December 2011. Strains were
systematically tested for 1st and 2nd line drugs, and
initially genotyped using spoligotyping. The subgroup
of strains collected from January 2005 to December
2011 was further studied by using 15 and 24 loci MIRuvNTR. Among the 1239 cases, 12.3% were resistant
and 2.1% MDR. A significantly higher proportion of
strains resistant to at least isoniazid (22.5%, p =
0.002) or rifampicin (20.0%, p <0.001) as well as
MDR (17.5%; p <0.001) was observed among relapse
cases than newly treated cases. Moreover, among the
fivegenotypicfamilies(T,30.1%-LAM,23.7%-H,
22.2% - EAI, 7.2% - X, 6.5%), 2 lineages, X and LAM,
were overrepresented among resistant and MDR
cases respectively. Our results showed that among
the19predominantspoligotypes,4weresignificantly
associated to drug resistance corresponding to
SIT 20, 64 (Family LAM), SIT 45 (Haarlem family)
and SIT 46 (unspecified family). Since, these ultraperipheral European departments are characterized
by sustained migration flows with mainland France
and surrounding countries among which some have
high TB incidence rates (Haiti 230/100 000; Guyana
111/100,000, Suriname 145/100,000), we further
investigated TB transmission among native and
foreign born patients in Guadeloupe (subgroup of
cases from 1 July 2006 to 30 June 2011). Results
obtained revealed that migrants in Guadeloupe had
an increased risk of developing TB with an incidence
rate seven times higher than in native subjects in
2010 (33.4 vs. 5.5 new cases/100,000 inhabitants).
These results will be discussed in the context of MIRu
typing results, and ongoing epidemiological and drugresistance surveillance studies as well a phylogenic
characterisation of M. tuberculosis isolates circulating
in the region.
P171
POPULATIONAL-BASED SURVEY OF
MOLECULAR AND EPIDEMIOLOGICAL LINKS
BETWEEN HUMAN AND ANIMAL INFECTIONS
BY MyCOBaCTERIUM BOvIS
Beatriz Romero1, Juan José Palacios2, Yurena
Navarro3, María Francisca Copano4, Lucía de
Juan5, Julio Alvarez6, Tatiana Alende1, Lucas
Domínguez5, Darío García de Viedma7
1
Madrid, Spain, CEI Campus Moncloa, UCM-UpM,
Madrid, Spain
2
CEI Campus Moncloa, UCM-UpM, Madrid, Spain
3
(VISAVET), Madrid, Spain; CEI Campus
Moncloa, UCM-UPM, Madrid, Spain; Servicio
de Microbiología Clínica y Enfermedades
Infecciosas. hospital general Universitario
Gregorio Marañón; Inst. Investigación Sanitaria
gregorio Marañón, Madrid, Spain
4
Laboratorio de Sanidad animal de Jove, gijón,
Spain
5
(Visavet), Madrid, Spain; CEI Campus Moncloa,
UCM-UPM, Madrid, Spain; Dpto. Sanidad AnimalFacultad de veterinaria, UCM, Madrid, Spain
6
(Visavet), Madrid, Spain; 9Inst. Ramón y Cajal de
Investigación Sanitaria (IRyCIS), Madrid, Spain
7
CEI Campus Moncloa, UCM-UpM, Madrid,
Spain; Servicio de Microbiología Clínica y
Enfermedades Infecciosas. hospital general
Universitario Gregorio Marañón; Inst.
Investigación Sanitaria gregorio Marañón,
Madrid, Spain; CIBER Enfermedades
Respiratorias (CIBERES), Spain
Mycobacterium bovis is the main causative agent
of tuberculosis (TB) in animals and few cases of
human tuberculosis due to this pathogen have been
reported in industrialised countries. In Spain, around
2% of the human TB cases are caused by M. bovis.
The prevalence of bovine tuberculosis in cattle
herds in Asturias (North Spain) is very low (0.14 in
2011) in comparison with the overall prevalence in
the rest of the country (1.33 in 2011). From 2006
to 2011 seventeen patients with M. bovis infection
were detected in Asturias. All corresponded to
Spanish-born cases, two were HIv-positive and the
average age was 61.4. In most cases existed the
possibility of a reactivation of an ancient TB infection
by immunosuppression (i.e age, HIv, respiratory
dysfunction). To challenge this hypothesis and to
evaluate potential recent transmission events we
carried out the molecular characterization of the M.
bovis isolates following a two-step scheme. Firstly,
human cases were genotyped by spoligotyping and
secondly, MIRu-vNTR was applied to the human
isolates together with all animal isolates (N=74) from
the farms located in their health-care area sharing
spoligotype with the human case. Genotyping of these
isolates showed eight (47%) human cases sharing
identical or highly similar genotype (differences in
one out of nine vNTR loci) with an animal isolate. A
coordinated epidemiological research was carried out
in order to support the molecular matches and direct
or indirect (farmer, livestock dealer, or consumers
of unpasteurized milk) relationships with animal
farms were found in five of them. Isolates from two
human cases living in the same city were clustered
between them, suggesting potential human-human
transmission. Our study constitutes a model of
integrative collaboration between human health
and veterinary efforts and means a snapshot of the
transmission dynamics of M. bovis in the humananimal interphase.
P175
RETROSPECTIVE ANALYSIS FOR DIAGNOSIS
OF MyCOBaCTERIUM TUBERCULOSIS
Huseyin Guducuoglu, Abdullah Bektas
Yüzüncü Yıl University School of Medicine
Medical Microbiology, van, Turkey
73
In this study aims to be whether suitability of samples
for diagnosis algorithm of Mycobacterium tuberculosis
in the last three years.
The samples obtained from June-2010 to March-2013
were submitted to the laboratory for diagnosis of
Mycobacterium tuberculosis. 1036 samples were
studied with the Ehrlich-Ziehl-Neelsen paint. various
culture methods such as Löwenstein-Jensen (LJ)
medium-BacT/ALERT® 3D and the BD BACTEC™
MGIT™ 320 Mycobacteria Culture System were
applied to the 508 samples. In addition, the real-time
PCR method was applied to the only 138 samples.
In general, for diagnosis of AFB, 2 or 3 sample were
ordered for each patient on the other hand only one
sample for each patient was ordered for culture. For
21 AFB positive samples, 13 cultures and 6 PCR
were positive, 5 culture and 1 PCR were negative. For
1015 AFB negative samples, 9 cultures and 6 PCR
were positive while 467 cultures and 103 PCR were
negative. Furthermore, 79 samples were examined
with PCR and culture method. For these samples, 3
were positive for culture and PCR, 78 samples were
negative culture and PCR, 2 samples were positive
for culture and negative PCR, and 6 samples were
identifiedasculture-negative,PCR-positive.
AFB and culture is likely to be inadequate to
determine Mycobacterium tuberculosis. Therefore,
PCR seems to be necessary. However it should be
also considered the clinical table of the patient to
decide whether PCR to be necessary or not. Hence,
the appropriate algorithm should be suggested to
diagnosis of Mycobacterium tuberculosis.
Coordinating the methods employed in the Member
States for diagnosing diseases; 2) Assisting actively
in the diagnosis of disease outbreaks in Member
States; 3) Facilitating the initial or further training of
experts in laboratory diagnosis with a view to the
harmonisation of diagnostic; 4) Collaborating with the
competent laboratories in third countries where those
diseases are prevalent; and 5) Conducting initial and
further training courses for the benefit of staff from
national reference laboratories and of experts from
developing countries.
Takingthesespecificresponsibilitiesandtasksinmind
the Eu-RL has designed an online database within
its website (www.bovinetuberculosis.eu) that allows
all the National Reference Laboratories to consult
the protocols used in other Member States in order
to facilitate harmonization of techniques throughout
the Community, in particular specifying standard test
methodologies. The Bovine Tuberculosis Protocols
Database compile all the information submitted from
alltheNRLstodefinethestateoftheartofthedifferent
methodologies (culture, extraction & identification,
molecular characterization and interferon gamma
detection) used all over Europe. This information is
evaluated by the Eu-RL and different activities are
performed (proficiency tests, evaluation of culture
media, determination of analytical sensitivity, etc.)
towards harmonization of protocols.
Moreover, among other activities, the Eu-RL gives
technical and laboratory support to all NRLs and send
reference reactives upon request (control strains,
DNA, DvR-spoligotyping membranes, etc.).
P176
P197
EUROPEAN UNION REFERENCE LABORATORY
FOR BOVINE TUBERCULOSIS: AN USEFUL
TOOL TOWARDS HARMONIZATION OF
PROTOCOLS
Beatriz Romero1, Javier Bezos1, Carmen Casal
Comendador1, Julio Alvarez2, Francisco Lozano1,
Nuria Moya1, Lucía de Juan3
1
Madrid, Spain
2
Madrid, Spain; Instituto Ramón y Cajal de
Investigación Sanitaria (IRyCIS), Madrid, Spain.
3
Madrid, Spain; Departamento de Sanidad
animal, Facultad de veterinaria, Universidad
Complutense de Madrid, Madrid, Spain
In 2008 the European union Reference Laboratory for
Bovine Tuberculosis (vISAvET Health Surveillance
Centre, Madrid, Spain) was designated with the main
activities (Commission Regulation 737/2008) of 1)
74
RAPID DIAGNOSIS, MOLECULAR TYPING
PATTERNS AND DRUG SUSCEPTIBILITY
PROFILES OF MYCOBACTERIA ISOLATED
FROM PATIENTS WITH TUBERCULOUS
MENINGITIS IN ANKARA
GÜLNUR TARHAN1, Hülya Şimşek2, Salih
CESUR3, Ismail CEYHAN4
1
Ahi Evran University, Kırşehir, Turkey
2
national public health agency, ankara Turkey
3
Etlik Training and Research hospital, ankara,
Turkey
4
4atatürk Chest Diseases and Thoracic Surgery
Training and Research hospital, ankara, Turkey
Background: Tuberculous meningitis (TBM) is
the most severe and lethal form of tuberculosis.
Conventional microscopy and culture has limited
utility for the paucibacillary nature of cerebrospinal
fluid(CSF)andtimeconsuming. Rapidandspecific
diagnosis of tubercular meningitis is of paramount
importance to decrease morbidity and mortality. In
this study, we aimed to evaluate rapid diagnosis of
tuberculous meningitis by COBAS Amplicor MTB and
Rotor Gene Real Time PCR, molecular typing and
drug susceptibility .
Methods: A retrospective study was conducted
on 468 patients of tubercular meningitis, tubercular
ascites and tubercular lymphadenitis in 7 years.
All specimens were evaluated smear microscopy,
Löwenstein Jensen culture and MGIT culture system.
COBAS Amplicor MTB and Rotor Gene Real Time PCR
were studied in accordance with the manufacturer’s
instructions. Culture positive specimens were
evaluated by spoligotyping for molecular typing.
Drug susceptibility patterns were determined using
conventional proportion method.
Results: using culture results as gold standard, the
sensitivity, specificity, positive (PPV) and negative
predictive values (NPv) of the COBAS Amplicor MTB
and Rotor Gene Real Time PCR were, repectively,
71%, 98.8%, 97.8% and 75% for COBAS Amplicor
MTB and 80%, 98.9%, 99% and 80 % for Rotor Gene
Real Time PCR. According to spoligotyping method, all
isolatesweredefinedasLAM7-TUR.Allisolateswere
found susceptible to isoniazid, rifampin,streptomycin
and ethambutol.
P198
POPULATION STRUCTURE OF
MyCOBaCTERIUM TUBERCULOSIS IN TWO
GEOGRAPHICALLY DISTANT AREAS OF
ARGENTINA
Johana Monteserin1, Ana Etchart2, Ana Reniero3,
Beatriz López1, Viviana Ritacco4
1
Instituto nacional de Enfermedades Infecciosas
argentina
2
hospital San Roque, Jujuy, San Salvador de
Jujuy, argentina
3
hospital Central, San Isidro, Buenos aires,
argentina
4
Instituto nacional de Enfermedadeinfecciosas
Argentina; National Council for Scientific
Research (COnICET), Buenos aires, argentina
Argentina is a large South American country with
diverse areas regarding physical geography, ethnic
composition and TB epidemiology. The cosmopolitan
area of Buenos Aires in the pampas presents medium
TB rates and receives migrants from highly endemic
TB areas of Argentina and neighbor countries. Jujuy,
an Andean province predominantly inhabited by
native Americans, presents one of the highest TB
rates in the country. We present an insight into the
Mycobacterium tuberculosis population structure in
those two areas which are 1,600 km distant from
each other.
We analyzed 383 isolates (one per patient): 158
were obtained in Jujuy in 2011-2012, and 225 were
collected in a Northern suburban district of Buenos
Aires in 2003-2007 and 2010-2012. Spoligotyping
was used for genotype assignation according to the
SITvIT database (www.pasteur-guadeloupe.fr:8081/
SITvITDemo).
In Jujuy the isolates displayed 56 different
spoligotypes. The family composition was: LAM 35%,
Haarlem31%,ill-definedT24%,S2%,andorphan
8%; the predominant shared types were SIT 50 H3
17%, SIT 33 LAM3 14% and SIT 42 LAM9 10%. In
Buenos Aires we found 79 different spoligotypes.
Frequencies of M. tuberculosis families were: LAM
37%,ill-definedfamilyT34%,Haarlem17%,S3%,U
2%, Beijing 1%, X 0.4%, and orphan 6%. Frequencies
in Buenos Aires were similar to those previously found
in a Western suburban district of the city. In Jujuy,
Haarlem strains were significantly more frequent
(p<0.0012), and T strains less frequent (p<0.015) than
in Buenos Aires. Of the 26 SIT-orphan strains found in
the study, 18 were grouped in 8 domestic clusters of
2-5 isolates. None of the SIT-orphan clustered strains
matched any of the >1000 spoligotypes from other
Ibero-American countries deposited in our database,
suggesting that those SIT-orphan genotypes might
be autochthonous.
The overall M. tuberculosis genotype distribution
observed in Argentina is in range with those reported
for other South American countries, with predominance
of LAM strains and local variations among the other
two major Euro-American families.
P214
PERFORMANCE OF SPOLIGOTYPING
IN ExTRAPULMONARY TUBERCULOSIS
DIAGNOSIS IN PATIENTS ATTENDING A
TERTIARY CARE HOSPITAL
Wellman Ribon1, Magda Lorena Orduz
Zambrano2
1
grupo de Inmunología y Epidemiología
Molecular, Universidad Industrial de Santander,
Colombia
2
Colombia; Master Student Biomedical Science,
Universidad Industrial de Santander, Colombia
Background: extrapulmonary tuberculosis cases
represent 15 to 20% of tuberculosis cases in
Colombia. Although, extrapulmonary tuberculosis
does not represent transmission risk, the patients
develop serious medical conditions. The diagnosis
of extrapulmonary tuberculosis is not easy by using
conventional methodologies such us smears and
culture because they are paucibacillary samples.
The development of molecular biology has allowed
the implementation PCR based test the diagnosis of
infectious diseases such as tuberculosis.
Objective: implementation of spoligotyping for
diagnosis of extrapulmonary tuberculosis in clinical
samples.
75
Methods: analysis of 63 samples of patients attending
a tertiary care hospital in Santander, Colombia which
were extrapulmonary tuberculosis suspected. Each
sample was processed in order to performe acid-fast
stains, the fluorescent auramine-rhodamine stain
and Ziehl-Neelsen stain, culture in Löwestein Jensen
medium and the DNA was extracted from with CTAB
method, following to amplify DR region by PCR and
hybridization by spoligotyping.
Results and discussions: the analyzed samples
corresponds a bone marrow aspirate (1/63), node
biopsy(1/63), liver biopsy (3/63), skin biopsy(1/63),
bloodculture(1/63),gastricfluid(3/63),cerebrospinal
fluid (26/63), ascitic fluid (3/63), urine(2/63), pleural
fluid(20/63), pericardial fluid(2/63). Of those 63
analyzed samples, 2 were positive for Ziehl-Neelsen
stain and 1 in Löwestein Jensen medium. For
spoligotyping 56 were positive for Mycobacterium
tuberculosis and 7 negative M. tuberculosis. The
morefrequentlysampleswerecerebrospinalfluidand
pleuralfluidandthefamiliesidentifiedforspoligotyping
were T1 (29/56), LAM9 (5/56), MANu2 (3/56), u (3/56)
and 14/56 were the patterns that are not reported
in the SpolDB4. Conclusion: The spoligotyping
is a good methodology that allows rapid diagnosis
of tuberculosis in extrapulmonary specimens, and
furthermore typing of the genotype of M. tuberculosis
involved in the infection, that contribute to molecular
epidemiology and control to disease.
Acknowledgments: Mycobacterium, Laboratorio de
Investigación y Extensión, Grupo de Inmunología y
Epidemiología Molecular, Maestría Ciencias Básicas
Biomédicas, universidad Industrial de Santander,
Colombia.
P219
GENETIC DIVERSITY OF MyCOBaCTERIUM
TUBERCULOSIS COMPLEx STRAINS ISOLATED
IN THE NORTH-WESTERN AND CENTRAL
COUNTIES OF ROMANIA
IONELA SORINA MUNTEAN1, Adriana Drăgan1,
Daniela Homorodean2, ANDREEA JODAL2, Sven
Hoffner3
1
pneumophtisiology hospital Brasov
2
national Reference Laboratory, Cluj-napoca,
Romania
3
Swedish Institute for Communicable Disease
Control, Solna; Supranational Reference
Laboratory Stockholm, Sweden
Background: Spoligotyping (spacer oligonucleotide
typing) is a genotyping technique that assesses
the genetic diversity of the direct repeat locus. It
is a fast and highly reproducible method useful for
clinical laboratory and molecular epidemiology of
tuberculosis.
Objectives: The assessment of the predominant
76
strain lineages among drug resistant strains of
Mycobacterium tuberculosis isolated from the NorthWestern and Central counties of Romania.
Material and methods: We studied 197 clinical
isolates of drug resistant strains of M.tuberculosis.
DNA was extracted by sonication and sent to the
Supranational TB Reference Laboratory Stockholm
for spoligotyping. The 197 strains originate from 14
counties of Romania. 180 (91.4%) were MDR-TB
strains, 13 (6.6%) were isoniazid mono-resistant
strains and 4 (2%) were rifampicin mono-resistant
strains.
Results: 179 (90.9%) from all the strains had a
shared international type number (SIT). They were
grouped in 11 clusters, the biggest one containing
alone 56 strains.The studied strains were identified
as belonging to the next families: Haarlem1 (56/197,
28.4%), T1 (30/197, 15.2%) T2 (23/197, 11.7%),
S (16/197, 8.1%), Haarlem 3 and Haarlem 4
-each(15/197, 7.6%), T3 (13/197, 6.6%). An amount
of18(9.1%)isolateswerenotidentifiedinSpolDB4
database. Strain families are distributed uniform in
the 13 counties.
Conclusions:
A. The high degree of clustering and the domination
of the Haarlem family among the tested strains.
B. No Beijing family strain was found among these
strains.
P220
MOLECULAR GENOTYPING CONFIRMS
TUBERCULOSIS INFECTION TRANSMISSION
IN HOUSEHOLD CONTACTS AS THE MAIN
INFECTION TRANSMISSION ROUTE AMONG
CHILDREN
Viesturs Baumanis1, Iveta Ozere2, Inta Jansone1,
Anda Nodieva2, Ilva Pole1, Matiss Bauskenieks1,
Girts Skenders2
1
Latvian Biomedical Research and Study Centre,
University of Latvia, Riga, Latvia
2
Riga East University hospital, Clinic of
Tuberculosis and Lung Diseases, Riga, Latvia
Tuberculosis (TB) incidence decreased from 74 per
100.000 population in 1998 till 36.2 in 2011 in Latvia,
however increased by 10% in 2012 including children
TB. Every month about 5 new cases of children
TB have been registered in spite of practically total
BCG vaccination and comparably low absolute
numbers of incidence. Therefore actual are studies
about infectious sources for chlidren TB which being
performed for prolonged time scale may clearly
characterise infection transmission and elaborate
protective measures. The goal of present study is
to analyse isolated form children Mycobacterium
tuberculosis (MT) genotypes with that ones of possible
infectious sources and analyse infection transmission
routes among children.
79 MT isolates (2000-2012) from children with
diagnosed TB and their possible infectious contacts
were analysed by IS6110 restriction fragment length
polymorphism and spoligotyping methods. Grouping
of RFLP`s was performed with BioNumerics software
using uPMG and Dice similarity. Found RFLP
patterns were compared with that of local database
and spoligotype in the SITvIT .
In 10% cases bacterial cultivation was successful
of clinically diagnosed TB only. Genotyping data in
comparison with possible infectious sources in 49
cases (62%) was identical, so confirming infection
transmission. Clustering rate was 40% in contrast
with the adult group where it is about 65% indicating,
that infection transmission in children collectives is not
actual. 10 (12%) of children isolates had no anologues
in that ones our data base, indicating on circulating
in the society of undiagnosed TB cases. Comparably
low was percentage of Beiging genotype, but multi
drug resistant isolates which among new cases is
the same than in adult group ~16%. In two cases
the spectrum fo drug resistance was stated to be
as extensive. Based on molecular genotyping of MT
solates analysis of family and social contacts clearly
demonstrarted prevalence of houshold infection
transmission among children and indicates directions
where protective measures should be concentrated
P227
WHOLE GENOME SEqUENCING REVEALS
LOCAL TRANSMISSION PATTERNS OF
MyCOBaCTERIUM BOvIS IN SYMPATRIC
CATTLE AND BADGER POPULATIONS
Roman Biek1, Anthony O‘Hare1, David Wright2,
Tom Mallon3, Carl McCormick3, Richard Orton1,
Stanley McDowell3, Hannah Trewby1, Robin
Skuce3, Rowland Kao1
1
University of Glasgow, Glasgow, Scotland, UK
2
queens University Belfast, Belfast, UK
3
agri-Food and Biosciences Institute, veterinary
Sciences Division, Belfast, UK
Whole genome sequencing (WGS) holds great
promise as a tool for studying the transmission
dynamics of an observed epidemic involving a
largely un-sampled ‘reservoir’ host, as is the case for
bovine tuberculosis (TB) in British and Irish cattle and
badgers. However, such data can require considerable
interpretation, particularly if used to infer patterns
of fine-scale transmission dynamics. Mathematical
models are ideal vehicles for this interpretation,
however approaches integrating models and genetic
data require good datasets to validate them. Here, we
analyse bovine TB transmission dynamics in Northern
Irish cattle and badgers, where there are extensive
demographic and livestock movement data, in
combination with M. bovis sequences from a spatially
clustered group of infected cattle and badgers.
Comparing WGS data to mathematical transmission
models showed good correlations between the
distributions of single nucleotide polymorphisms and
the spatial and within-herd contact structure, but poor
correspondence to the network of cattle movements
linking them. Despite badgers being under-sampled,
our data provided evidence for recent, ongoing
transmission events between the two host species,
and an unprecedented resolution of the spatial
correlation between these. These results provide the
first direct genetic evidence of M. bovis persistence
on farms over multiple outbreaks with a continued,
ongoing interaction with M. bovis in badgers. There
was also genetic evidence consistent with cattlecattle transmission in some of the study herds.
P230
MULTIDRUG RESISTANT TUBERCULOSIS
EPIDEMIC IN SWAZILAND: ONGOING
TRANSMISSION OF MULTIDRUG RESISTANT
MyCOBaCTERIUM TUBERCULOSIS STRAINS
M. Merker1, E. Sanchez2, P. Beckert1, F. Jochims3,
M. Bonnet2, Th. Dlamini4, M. Bastard2, H.
Koarakozian3, S. Rüsch-Gerdes1, S. Niemann1
1
2
Epicentre, paris, France
3
Medecins Sans Frontieres,geneva, Switzerland
4
national Tuberculosis Control programme,
Mbabane, Swaziland
Emergence and effective transmission of multidrug
resistant tuberculosis (MDR-TB) strains have been
reported in several countries worldwide and threaten
local and global TB control. Especially in Sub-Saharan
Africa the TB epidemic has been accelerated by both,
high HIv/TB co-infection rates and the emergence of
MDR strains. To avoid a wide unrecognized spread of
MDR Mycobacterium tuberculosis complex (MTBC)
strains, it is necessary to systematically monitor
resistancelevelsanddeterminefactorsinfluencingthe
MDR epidemic in several Sub-Saharan countries.
To address this question, we performed a national
drug resistance survey in 2009-2010 in Swaziland,
a small kingdom within South Africa with the world’s
highest HIv/TB prevalence. Screening of 988 patients
revealed MDR-TB rates of 7.7% and 33.8% among
new cases and previously treated cases, respectively.
The high level of MDR TB in new cases already
indicated ongoing transmission of MDR-TB within
the population and an urgent need for strengthening
diagnostics and treatment facilities.
To further investigate recent transmission levels and
association between population structure and MDRTB, 412 isolates including all drug resistant strains
were investigated by genotyping (24-loci MIRu-vNTR
typing and spoligotyping) and Sanger sequencing
of known resistance conferring genes (katG, inha,
77
rpoB, embB, pnca). The population structure was
dominated by strains of the MTBC belonging to the
S-type (8.6%), X-type (15.5%), LAM (23.3%) and
Beijing lineage (23.7%). Furthermore, we determined
high clustering rates among MDR strains that were
confirmed by particular profiles of drug resistance
mutations. In the multivariate analysis, MDR-TB was
associated with S-type (OR 4.02), HIv infection (OR
2.86), female gender (OR 1.98) and clustering (8.70).
Nor Beijing strains (OR 0.06) neither LAM strains (OR
0.12) were associated.
In conclusion, our study identified unrecognized
transmission of MDR strains as a major driver
of the MDR epidemic in Swaziland. These data
emphasize the need for an ongoing control of
MDR-TB transmission in MDR-TB/HIv high burden
countries like Swaziland, to evaluate the success and
limitations of newly introduced diagnostics tools, e.g.
the Xpert MTB/RIF and to prevent the expansion of
highly transmissible MDR MTB clones.
P231
TB-MINER, AN ON-LINE TOOL TO DESCRIBE
CLASSIFICATION OF M. TUBERCULOSIS
COMPLEx BASED ON THE WIDE SCALE
DIVERSITY OF THE NETHERLANDS DATABASE
Jérôme Azé1, Guislaine Refrégier2, Kristin
Kremer3, Dick van Soolingen4, Christophe Sola2
1
Université paris-Sud, Laboratoire de Recherche
En Informatique, Orsay, France
2
Et Microbiologie, Infection genetics Emerging
3
Who Regional Office for Europe; Tuberculosis
and M/XDR-TB programme, Copenhagen,
Denmark
4
national Institute for public health and
the Environment, Bilthoven, Netherlands;
Department of Medical Microbiology, Radboud
University nijmegen Medical Centre, nijmegen,
netherlands
This study shows the synergism between knowledge
discovery using data (KDD) methods and high
throughput genomics (24 vNTR loci and spoligotyping
analysis). By using Weka® on the Dutch tuberculosis
genotyping database, we increased our understanding
of Mycobacterium tuberculosis complex (MTC)
taxonomy and epidemiology. We propose 14
spoligotype-based larger genotype families; M.
africanum 1, M. africanum 2, Beijing, M. bovis, CAM,
CAS, EAI, H, LAM, S, T, TuR, ural and X as compared
tothebroaderclassificationin6lineageofGagneux
et al. To share this knowledge, we provide an online tool; TBminer, that classifies genotypes based
on both spoligotyping and vNTR analysis, and that
summarizes key molecular epidemiological data.
In total 3454 clinical isolates were retrieved from as
78
many patients diagnosed with tuberculosis in the
Netherlands in the period 2004-2008. Genotyping
was performed using: (1) 24-vNTR run on capillary
sequencers, (2) spoligotyping run on Luminex®
200, (3) IS6110-RFLP. vNTR typing produced the
highest degree of discrimination. Spoligotyping was
confirmed a very powerful classifier in genotype
families. To balance potential convergence events
in spoligotyping, we also tested a combination of
induction algorithms relying on 24 vNTR data and
14 families classification mentioned above. Most
non-matching genotypic data concerned isolates
classified as T or LAM already pinpointed for their
lower phylogenetic relevance.
As the number of genotyping techniques expands
for each pathogen, this type of approach could be
transferred to other pathogens to take best advantage
of all available information.
P241
DISTRIBUTION AND SPATIAL ANALYSIS OF
FOCI OF TUBERCULOSIS IN CATTLE AND
DEERS IN THE SOUTH OF BEIRA INTERIOR
(PORTUGAL)
Luis Caiola Ribeiro1, Paulo Fernandez2, Manuel
Martins2
1
general Direction of Feed and veterinary,
portugal
2
School of agriculture, polytechnic Institute of
Castelo Branco, Castelo Branco, portugal
Bovine Tuberculosis (BT) disease is an animal with
high economic impact. The etiologic agent of this
disease is the bacteria Mycobacterium bovis which,
in addition to affect cattle, also causes tuberculosis in
other mammalian species, such this in study: Cattle
(Bos taurus), Red Deer (Cervus elaphus), including
humans and are thus included in the category of
zoonotic diseases.
The transmission of infectious disease is closely
related to the concepts of spatial and space-time
proximity, so that the transmission will have a greater
probability of the larger sharing these concepts by
the individuals at risk. The epidemiological analysis
should take into account the two concepts.
The Geographic Information Systems (GIS) are
now considered an essential and valuable tool in
epidemiology. The analysis of spatial visualization,
exploration and modeling allows a more thorough
knowledge about the spatial and temporal dynamics
of the disease, and can be used for suggesting and
support new epidemiological hypotheses.
The use of GIS technologies (Average Nearest
Neighbor, Global Moran I, Local Moran I (LISA),
Getis-Ord Gi* Statistics (Hot-Spots) and Ellipse
Standard Deviation (Directional Deviation)) analyze
and describe spatial patterns of distribution of BT in
parishes of nUtS III: Beira Interior Sul (Portugal)
The results revealed during the study period (20012010), an average prevalence of Bovine Tuberculosis
in cattle for mainland Portugal is 0.10%, for Beira
Interior is 0.09% and 0.42% in Beira Interior Sul.
For the average prevalence in farms was 0.29% in
Portugal, 0.24% in nUtS II: Beira Interior and 2.37%
in nUtS III: Beira Interior South.
The results will lead to support decision-making in
relation to cattle and their handling, and hunting and
repopulation of wild ungulates.
A spatial statistical analysis applied to counties with
outbreaks of TB in cattle was detected clusters and
correlations in two parishes (Castelo Branco and
Rosmaninhal) and dispersion in two others (Monforte
and Monfortinho). The spatial autocorrelation (Global
and Local) sensed distances between 13 and 25 km
as being those where the grouping is more intense,
and regarded as Hot Spot areas of the parishes of
Malpica do Tejo, Monforte, Rosmaninhal, Ladoeiro,
Segura and Zebreira.
The dispersion or clustering of outbreaks may be
related to the proximity of the farms, with the scattering
properties, with the exchange of animals between
farms of the same owner, buying and selling animals
or with health problems (s) Producer (s).
Regarding the activity of hunting, the beats and
mounts can lead to deers are “pushed” to more remote
locations, which increase their dispersal, which
increases the likelihood of spread of pathogens.
The proximity to Spain may have some relationship
intheareasofinfluenceofhotspotsdeterminedby
spatial statistics, since hunting wild animals roam
freely and with ease between the two states.
P242
TUBERCULOSIS IN THE CZECH REPUBLIC
AT RISK GROUPS: CASUISTRY FROM THE
CURRENT TIME
Maria Müllerová , M. Vašáková , E. Kopecká , J.
Homolka2, J. Pohl2, K. Křepela2
1
Laboratory of Mycobacteriology, Citylab Ltd.,
prague, Czech Republic
2
Department of pneumology, prague, Czech
Republic
1
2
2
Tuberculosis is still an important infectious disease,
as old as humanity itself. In the Czech Republic
there is an ongoing trend of declining incidence of
TB, including TB in children. In November 2010 the
area-wide vaccination of children ended and in 2012
it was switched to selective vaccination of groups atrisk. The Czech Republic lying in the heart of Europe
is a crossroad for migrants and immigrants bearing
risk of importing tuberculosis, including its MDR-TB
form. The most important risk group are foreigners
from countries with high TB incidence, often with
MDR-TB, the treatment of which is time-consuming
and costly. Other risk groups are homeless people,
squatters, unemployed people, alcoholics, but also
people under stress and work strain. We present
the following case reports of TB patients from highrisk groups from recent time period: 1) unvaccinated
child of mixed marriage, a Czech mother, a father
from Africa, treated for TB process. Child inspected
ascontactwithTBfather.TBforchildconfirmedby
X-ray and also by gastric lavage and BAL: BACTEC
MGIT 960 and cultivation positive, MTD test probable.
2) A boy from China, without proof of vaccination,
grandfathertreatedinChinaforlungTB.TBconfirmed
by X-ray and from sputum: cultivation, BACTEC
MGIT 960 and quantiFERON positive, MTD test
probable. 3) An unemployed man from ukraine. TB
confirmed from sputum: microscopy, cultivation and
MTD test positive. 4) Female from Kyrgyzstan. TB
confirmedbyX-ray,cultivationandMTDtestpositive.
5) An unemployed man, alcoholic, living in squat,
infected his roommate (contact). Sputum of both
men: microscopy, cultivation and MTD test positive.
6) A student with TB infected his younger brother
(contact). They both live in a “normal” functioning
family, students, since childhood racing sportsmen.
Sputum from both: microscopy, cultivation, BACTEC
MGIT 960 and MTD test positive. Conclusions: Early
diagnosis of new cases, prompt investigation of
contacts, proper treatment, supervision of patients,
taking into account the patient’s country of origin, and
not forgetting selective vaccination of children from
risk groups. This all helps to keep the favorable trend
of TB incidence in the Czech Republic.
P250
CORRELATION BETWEEN STREPTOMYCIN
INTERMEDIATE-LEVEL RESISTANCE AND
GIDB MUTATION IN AN ENDEMIC MULTIDRUGRESISTANT TUBERCULOSIS CLUSTER
Joao Perdigao1, Rita Macedo2, Diana Machado3,
Carla Silva1, Luisa Jordao4, Isabel Couto3, Miguel
Viveiros3, Isabel Portugal1
1
Uria, Centro de patogénese Molecular,
Faculdade de Farmácia Da Universidade de
Lisboa, Lisbon, portugal
2
public health Laboratory: Micobacteriology/
Tuberculosis, public health Department,
administração Regional de Saúde de Lisboa E
vale Do Tejo, I.p., Lisbon, portugal
3
Microbiologia Médica, Instituto de higiene e
(Ihmt, Unl), Lisbon, portugal
4
Departamento de Doenças Infecciosas, Instituto
nacional de Saúde Dr. Ricardo Jorge, Lisbon,
portugal
Development
of
streptomycin-resistance
in
Mycobacterium tuberculosis is usually associated
with mutations in rpsl and rrs genes, although up to
79
50% of clinical streptomycin-resistant isolates may
present no mutation in either of these genes. The
situation in Lisbon Health Region is similar, although
mutations in rrs gene are only rarely detected. In the
present report we investigate the role of gidB gene
mutations in streptomycin resistance.
We have analyzed 52 streptomycin-resistant and 30
streptomycin-susceptible Mycobacterium tuberculosis
clinical isolates by sequencing and endonuclease
analysis of the gidB and rpsl genes. All clinical
isolates were genotyped by 12-loci MIRu-vNTR.
Semiquantitative drug susceptibility testing was also
performed to a select set of isolates to assess the
resistance levels towards streptomycin.
The gidB gene of 18 streptomycin-resistant isolates
was sequenced and four missense mutations were
found: F12L (1/18), L16R (18/18), A80P (4/18) and
S100F (18/18). The remaining isolates were screened
by endonuclease analysis for mutations A80P in gidB
and K43R in rpsl gene. Overall, mutation A80P in
gidB gene was found in 7 streptomycin-resistant
isolates and 12 streptomycin-susceptible multidrug
resistant isolates. Also noteworthy, comparison of the
distribution of gidB, rpsl and rrs mutations revealed
that gidB A80P mutation was only present in isolates
without rpsl and rrsmutations.Moreover,thisspecific
mutation was found among all isolates belonging to
genetic cluster q1.
Streptomycin quantitative drug susceptibility testing
showed that isolates carrying the GidB A80P mutation
were streptomycin intermediate-level resistant and
that standard drug susceptibility testing yielded
inconsistent results probably due to borderline
resistance.
Bioinformatic analysis on the degree of conservation
showed that the GidB A80P mutation is predicted to
affect protein function.
We conclude that gidB mutations may explain the
high number of streptomycin-resistant strains with
no mutation in rpsl or rrs. These mutations might
occasionally confer undetected streptomycin lowlevel resistance in regular drug susceptibility testing.
Also, GidB A80P mutations may serve as surrogate
markers for q1 cluster isolates that are associated with
multidrug/extensively drug-resistant tuberculosis.
P257
DANISH MyCOBaCTERIUM TUBERCULOSIS
OUTBREAK STRAIN IS SPREADING AMONG
GREENLANDIC INUIT IN DENMARK AND
GREENLAND
Troels Lillebaek1, Ase Bengard Andersen1, Erik
M. Rasmussen1, Zaza Kamper-Jørgensen1,
Matthias K. Pedersen1, Karen Bjoern-Mortensen2,
Karin Ladefoged2, Vibeke Ø. Thomsen1
1
Statens Serum Institut, Copenhagen, Denmark
2
Dronning Ingrids Sundhedscenter, nuuk,
greenland
80
Transmission
of
Mycobacterium
tuberculosis
continues at high rates among Greenlanders in
Greenland and Denmark, with 203 and 450 notified
cases per 105 populations year 2010, respectively.
We can document, that the predominant Danish
M. tuberculosis outbreak strain “C2/1112-15” has
been transmitted to Greenlanders in Denmark, and
subsequently to Greenland, where it is spreading
at alarming rates, adding to the already heavy
tuberculosis burden in this population group. It is
now clear, that “C2/1112-15” is able to multiply in
genetically very different populations. Thus, it might
have the ability to spread even further keeping in
mind the potential clinical consequences of strain
diversity, e.g. the widely spread Beijing genotype.
The introduction of the predominant C2/1112-15 M.
tuberculosis strain into the Inuit community in the
Arctic Circumpolar Region is an alarming tendency
which deserves attention. We need to monitor whether
this strain already have, or will, spread outside The
Danish Kingdom.
P258
ACTIVE TRANSMISSION OF MyCOBaCTERIUM
TUBERCULOSIS (MT) CONTINUES AT
SURPRISINGLY HIGH RATES IN DENMARK
Troels Lillebaek1, Åse Bengård Andersen1,
Niels Jørgen Seersholm2, Vibeke Østergaard
Thomsen1
1
Statens Serum Institut, Copenhagen, Denmark
2
Copenhagen University hospital gentofte,
Copenhagen, Denmark
Active transmission of Mycobacterium tuberculosis
(Mt) continues at surprisingly high rates in Denmark
(DK).Itisseeninspecifichighrisksegments
of the population with social problems such as
homelessness, alcohol, and/or drug abuse. The
patients are infected with the Danish “C”/1112-15” Mt
outbreak strain, and the transmission is attributed to
delayed diagnosis. The present situation demands
increased focus on early tuberculosis diagnosis and
control of transmission, and improved actions calls
for prioritizing the area politically and economically.
MDR DIAGNOSTIC AND DST
P15
RESISTANCE PATTERNS TO SECOND-LINE
DRUGS IN MyCOBaCTERIUM TUBERCULOSIS
IN SPAIN
Pilar Ruiz1, Juan B Gutierrez1, Manuel Causse1,
Manuel J Casal 1
1
University of Córdoba, Córdoba, Spain
Introduction: The emergence of TB resistant to both
first- and second-line drugs (XDRTB) (XXDRTB) is
a major cause of concern, particularly because it is
associated with elevated mortality. Spain has one of
the highest TB rates in Western Europe.
Aims: This study sought to investigate secondline drug resistance in Mycobacterium tuberculosis
strains isolated at the university Hospital “Reina
Sofía”(Córdoba, Spain) from 2006 to2011 as well as
in strains kept at a Mycobacterial Reference Center.
The most common mutations conferring resistance to
thesecond-linedrugswerealsoidentified.
Material and Method: Of the total 946 Mycobacterium
tuberculosis strains analysed, 393 were isolated
from 26,183 specimens obtained from patients with
clinically-suspected tuberculosis at the university
Hospital “Reina Sofía” (Córdoba, Spain) between 2006
and 2011. The remaining 553 strains were referred
by various hospitals to the Mycobacterial Reference
Center (from an area with 8,240,400 habitants)
Drug susceptibility was tested using the BACTEC
MGIT 960 automated mycobacterial detection
system.Phenotypic resistance was confirmed using
the Genotype MTBDR plus test to detect resistance to
RIF ( mutations in gene rpoB) and to INH ( mutations
in genes inhA and KatG). Strains were genotyped
using the Genotype MTBDRsl test, to detect mutations
conferringresistancetofluoroquinolones(genegyrA,
encoding the DNA gyrase enzyme), aminoglycosides/
cyclic peptides (16S rRNA gene, rrs) and ethambutol
(gene embB).
Results: Of the 946 strains examined, 156 (16.49%)
displayed resistance to at least one first-line drug,
while 51 strains (5.39%) were resistant to at least
one second-line drug. Multidrug resistance (MDR)
was detected in 39 strains (4.12%). Three pre-XDR
strains (0.31%) and three XDR strains (0.31%) were
detected. The greatest resistance of MDR strains was
to rifabutin (35.89%), followed by rifapentine (30.76%),
ethionamide (30.76%), capreomycin (12.82%),
ofloxacin (10.25%) and kanamycin (2.56%). Most
MDR strains resistant to second-line drugs displayed
the pattern: RIF+INH+RFB+RFP.
The most important patterns of resistance were RIF+I
NH+STR+EMB+PZA+CAP+KAN+ETH+OFX+RFB.
In strains tested for GyrA mutations associated
with fluoroquinolone resistance, the most common
mutation was S91P (35.7%), followed by A90v and
D94N/y (21.4%). The D94G mutation was found
in 7.1% of strains. Testing for rrs gene mutations
associated with resistance to AMK/KAN/CAP detected
the A1401G mutation in all resistant strains.
Conclusions: The resistance detected here to
second-line anti-TB drugs confirms the presence
ofanewchallengeinthefightagainsttuberculosis.
Epidemiological surveillance of resistance to anti-TB
drugs based on phenotype and genotype analysis is
essential.
P27
EVALUATION THE BACT ALERT 3D SYSTEM
FOR RECOVERY AND IDENTIFICATION OF
MYCOBACTERIA FROM CLINICAL SAMPLES
María Rosarys Martínez Romero1, Misleidis
Sardiña2, Grechen García2, Lilian María Mederos2
1
pedro Kourí Institute, havana, Cuba
2
national Reference Laboratory in Tuberculosis
and Mycobacteria, pedro Kourí Institute, havana,
Cuba
Background: The rapid diagnosis of M. tuberculosis
is essential to implement the adequate antimicrobial
therapy and effective control of this disease.
Aim: To evaluate a BacT ALERT 3D automatic system
for mycobacteria isolates.
Methods: Were study 819 clinical samples received
at the National Reference Laboratory in Tuberculosis,
IPK, from August 2010 to November 2011. The
samples were inoculated in parallel in Löwenstein
Jensen medium and BacT ALERT 3D automatic
system. The results obtained were analyzed and
compared by number of isolates, time detection of
growth (TDG), contamination rate (CR) and calculated
the quality indicators of BacT ALERT 3D automatic
system.
Results: By Löwenstein Jensen (LJ) was obtained 99
(90%) positives isolates, 102 (92.7%) by BacT ALERT
3D and 89 (80.9%) by two methods simultaneously.
The TDG average of BacT ALERT 3D was 16.67
days and 22.50 days for LJ. The CR was 7.4% and
6.7 % for LJ and BacT ALERT 3D, respectively. The
concordance between BacT ALERT 3D and LJ was
97.82%.Thesensitivity,specificityandYoudenindex
obtained by BacT/ALERT 3D was 97.75%, 98.44%
and 0.92, respectively.
Conclusions: BacT/ALERT 3D system is a
suitable method for recovering mycobacteria from
81
clinical samples. It demonstrated a shorter TDG of
mycobacteria than LJ medium which was very useful
to start with antimicrobial therapy in patient’s negative
smear with human immunodeficiency virus. The LJ
culture it must be used in combination with automatic
systems for to assure a total mycobacterial recovery.
P28
DETECTION OF AMIKACIN RESISTANCE IN
MyCOBaCTERIUM TUBERCULOSIS USING
THE NITRATE REDUCTASE ASSAY AND THE
RESAZURIN MICROTITRE ASSAY
Dihadenys Lemus1, Carlos Washington Reyes2,
Miguel Echemendia1, Juan Carlos Palomino3,
Anandi Martin3
1
Institute of Tropical Medicine pedro Kourí,
havana, Cuba
2
Laboratorio provincial de Diagnóstico de
Micobacterias de Santa Elena. Instituto nacional
de Investigación En Salud pública, Santa Elena,
Ecuador
3
ghent University, ghent, Belgium
Background: Amikacin (AMK) is one of the
recommended second-line drugs for multidrug
resistant (MDR) tuberculosis (TB) treatment. The
recommended methods for AMK susceptibility testing
are BACTEC-460 and MGIT-960, both based in
growth in liquid media, but their implementation are
prohibitive in low-resource countries. The aim of
this study was to develop two alternative methods,
nitrate reduction assay in liquid media (L-NRA) and
resazurin microtitre assay plate (REMA), to investigate
resistance to AMK in Mycobacterium tuberculosis.
Methods: Thirty-one M. tuberculosis strains
previously studied by the MGIT-960 were used
to validate L-NRA and REMA and then they were
applied in 29 M. tuberculosis MDR clinical isolates
from Cuba. L-NRA was carried out in Middlebrook
7H9 medium suplemented with OADC (7H9-S) with
critical concentration of 1 µg/mL of AMK. REMA was
performed in sterile 96-well plate using 7H9-S; AMK
range used was 8-0.25 µg/mL. For MDR isolates the
rrs gene was investigated by the Genotype MTBDRsl
assay.
Results: Both, the L-NRA and REMA, showed
sensitivities of 100% and specificities of 80.0%.
The 81.8% of M. tuberculosis MDR isolates with rrs
gene wild type pattern, by the Genotype MTBDRsl
assay, were susceptible to AMK by both phenotypic
methods;sevenisolateswereclassifiedasresistant
by the molecular assay, 85.7% of them had a minimal
inhibitory concentration of 8 µg/mL and 71.4% were
resistant by the L-NRA.
Conclusions: This study shows a high level of
agreement of these two low-cost methods for AMK
resistance detection. Further standardization is
requiredtoimprovetheirspecificities.
82
P37
GENOMIC MUTATION PROFILING OF
MULTIDRUG RESISTANCE TUBERCULOSIS
ISOLATES USING BY NOVEL GENOTYPE®
MTBDRPLUS ASSAY
Anand Kumar Maurya1, Surya Kant2, Amresh
Kumar Singh3, Ram Awadh Singh Kushwaha2,
Manoj Kumar3, Vijaya Lakshmi Nag3, Tapan N
Dhole3
1
Department of pulmonary Medicine, King
George’s Medical University, Lucknow, India;
Department of Microbiology, Sanjay gandhi
post graduate Institute of Medical Sciences,
Lucknow, India
2
Department of pulmonary Medicine, King
george’s Medical University, Lucknow, India
3
Department of Microbiology, Sanjay gandhi
postgraduate Institute of Medical Sciences,
Lucknow, India
Objectives: The emergence and spread of multidrugresistant tuberculosis (MDR-TB) of Mycobacterium
tuberculosis poses a significant threat to the global
control of tuberculosis. Rapid detection of MDR-TB
allows the establishment of an effective treatment
regimen; minimizes the risk of further resistance
and limits spread of drug resistant strains. The aim
of the study was to rapid detection and genomic
mutations profiling of rpoB, katG and inhA genes in
clinical isolates of MDR-TB by the novel GenoType®
MTBDRplus assay in Northern India.
Methods: We conducted a prospective study and
five hundred fifty specimens were collected from
highly suspected of drug resistance of pulmonary
tuberculosis and extra pulmonary tuberculosis cases
from January 2011 and January 2013; which was
processed by Ziehl-Neelson (ZN) staining, culture,
differentiation by the GenoType®CMassay,firstline
drug susceptibility test (DST) using BacT/ALERT
3D system and GenoType® MTBDRplus assay for
performance, frequency and genomic mutation
patterns of MDR-TB.
Results: Among a total of 550 specimens collected
from 423 (76.9%) PTB and 127 (23.1%) EPTB patients
of highly suspected cases of treatment defaulters, retreatment and relapse cases, only 103 (18.7%) were
AFB positive in ZN microscopy and 257 (46.7%) were
positive for mycobacteria by BacT/ALERT 3D system.
Among a total of 209 MTBC strains, readable results
were obtained from 206 (98.5%) MTBC strains by
GenoType® MTBDRplus assay. The sensitivity and
specificity of the GenoType® MTBDRplus assay for
RIF, INH and MDR-TB strains were 98.0 % (95% CI:
92.88 % - 99.70 %); 98.8 % (95% CI: 93.59 % - 99.80
%), 98.4 % (95% CI: 94.24 % - 99.76 %); 98.8% (95%
CI: 93.67 % - 99.81 %) and 98.2 % (95% CI: 90.24
% - 99.70 %); 100.0 % (95% CI: 97.56 % - 100.00
%). Among a total of 55 MDR-TB strains, 45 (81.8%),
52 (94.5%), and 17 (30.9%) strains harbored known
mutation in rpoB, katG and inhA genes respectively.
Conclusions: Our study demonstrated the most
prominent mutations in rpoB, katG and inhA genes
were 37 in S531L (67.3%), 52 in S315T1 (94.5%),
and 11 in C15T (20%) region respectively (p <0.05).
We found transmission of prominent mutations is
contributing to an unexpected increase in primary
resistance, including MDR TB cases in Northern India.
GenoType® MTBDRplus assay is a high sensitive,
short turnaround, and rapid test for the detection of
MDR-TB in northern region of India.
Keywords: GenoType® MTBDRplus assay, MDR-TB,
M. tuberculosis complex, Tuberculosis.
P69
FIELD EVALUATION OF THE DIRECT
COLORIMETRIC NITRATE REDUCTASE ASSAY
FOR THE SIMULTANEOUS DETECTION OF MDRAND xDR-TB IN ARGENTINA
Belen Imperiale1, Nora Morcillo1, Juan Carlos
Palomino2, Anandi Martin2
1
hospital Dr. Cetrángolo, Tuberculosis Control
program Reference Laboratory, Buenos aires,
argentina
2
Laboratory of Microbiology, Department of
Biochemistry & Microbiology, ghent University,
ghent, Belgium
Objectives: The spread of MDR-TB and XDR-TB
stresses the need for quick and affordable diagnostic
tools, particularly for low- and middle-income
countries, with a high prevalence of TB. The objective
of this study was to evaluate the previously described
Nitrate Reductase Assay for the simultaneous
detection of MDR and XDR-TB patients directly on
sputum samples (D-NRA).
Methods: The performance of D-NRA was compared
with the BACTEC MGIT960 assay under routine
conditions. Sputum smear-positive samples were
decontaminated with the NALC method and inoculated
in Lowenstein-Jensen tubes containing drugs and 1
mg/ml of potassium nitrate. The following drugs were
tested: rifampicin (RMP), isoniazid (INH), ofloxacin
(OFLO), kanamycin (KAN). Consecutive smearpositive samples (n= 92) were prospectively analysed
with D-NRA and compared to BACTEC MGIT960 to
detect resistance to INH, RMP, KAN and OFLO. PNB
was included for the simultaneous identification of
Mycobacterium tuberculosis complex.
Results: Out of 92 smear-positive samples, 6 failed
to growth and 2 were identified as non-tuberculosis
mycobacteria. For the 84 samples that grew, the NRA
sensitivity,specificity,positiveandnegativepredictive
values for all drugs were 100%. The mean time to
obtain results with the D-NRA was 13.9 days and the
overall agreement between D-NRA and the BACTEC
MGIT960 was 100% for all drugs.
Conclusion: D-NRA offers timely drug resistance
detection of M. tuberculosis resistance to first- and
second-line drugs. D-NRA was user-friendly, easy to
implement in limited laboratory facilities and provide
an easy interpretation of results by a simple visual
change of color.
P80
EVALUATION OF THE AID TB RESISTANCE
LINE PROBE ASSAY FOR RAPID DETECTION
OF GENETIC ALTERATIONS ASSOCIATED WITH
MyCOBaCTERIUM TUBERCULOSIS DRUG
RESISTANCE
Claudia Ritter1, Katja Lucke4, Frick Sirgel3, Robin
Warren3, Erik Böttger1, Guido Bloemberg1
1
2
nationales zentrum für Mykobakterien, zürich,
Switzerland
3
DST/nRF Centre of Excellence for Biomedical
TB Research/MRC Centre for Molecular and
Cellular Biology, Division of Molecular Biology
and human genetics, Faculty of health Science,
Stellenbosch University, Cape Town, South
africa
4
Universität Zürich, Zürich, Switzerland; Unilabs
zurich, zürich, Switzerland
Rapid detection of drug resistance mutations
facilitates implementation of adequate therapy and
limits spread of multi-resistant M. tuberculosis strains.
The AID TB Resistance line probe assay (Autoimmun
Diagnostika GmbH, Strassberg, Germany) offers
screening for the most prevalent mutations confering
resistance to isoniazid, rifampicin, streptomycin,
kanamycin,amikacin,capreomycin,fluoroquinolones
and ethambutol. using clinical M. tuberculosis isolates
from low and high endemic areas (Switzerland, n=110;
South Africa, n=67), the line probe assay detected
with a 100% accuracy resistance mutations for which
oligonucleotide probes are present in the assay.
Subsequently, the line probe assay was shown to
allow for rapid assessment of drug resistance in early
positive broth cultures.
Finally, the line probe assay was analysed for direct
screening of smear positive clinical specimens.
Screening 98 clinical specimens demonstrated
that the test gave an interpretable result in> 95%.
Antibiotic resistance mutations detected in the clinical
sampleswereconfirmedbynucleicacidsequencing
and phenotypic testing of the corresponding culture
isolates. A 100% agreement between the results
of molecular screening of smear positive clinical
specimens with that of drug susceptibility testing of
the corresponding culture isolates was observed. We
conclude that the TB Resistance line probe assay
(AID) is an accurate tool for the rapid detection of
resistance mutations in cultured isolates and in smear
positive clinical specimens.
83
P85
A NEW COLORIMETRIC PLATE FOR THE
RAPID DIAGNOSIS OF ExTENSIVELY DRUGRESISTANT TUBERCULOSIS DIRECTLY IN
SPUTUM SAMPLES
Beatriz Lopez1, Lucia Barrera1, Martha
Ambroggi2, Susana Poggi2, Peter Vandamme3,
Juan Carlos Palomino3, Viviana Ritacco1, Anandi
Martin3
1
Instituto nacional de Enfermedades Infecciosas
argentina
2
Laboratorio de Micobacteriología, hospital F. J.
Muñiz; CONICET, Buenos Aires, Argentina;
3
Laboratory of Microbiology, Department
of Biochemistry and Microbiology, ghent
University, gent, Belgium
Objectives: The nitrate reductase assay (NRA)
assay was recently approved by WHO for rapid
rifampicin and isoniazid susceptibility testing of
Mycobacterium tuberculosis. A modification of the
technique is proposed, which consists in employing
a multi-well plate format and enriched agar medium.
The objective of this study is to evaluate the plateNRA directly on sputum samples (D-NRA) for early
detection of extensively drug-resistant (XDR-TB).
Methods: This preliminary study is currently
performed in Buenos Aires, Argentina. Smearpositive sputum samples from 26 consecutive TB
patients at risk of treatment failure were assayed.
Sputa were decontaminated with NALC-NaOH.
Rifampicin,isoniazid,ofloxacin,kanamycin,amikacin
and capreomycin were tested at 1, 0.2, 2, 6, 2, and
5 µg/ml, respectively, in 24-well plates containing
Middlebrook 7H11-KNO3 agar. P-nitrobenzoic acid
(PNB) was used for differentiating M. tuberculosis
from other mycobacteria, and PRa-hsp65 was used
asspeciesidentificationreferencemethod.Accuracy,
turnaround time and cost were evaluated using the
MGIT 960 system as gold standard method.
Results: Out of 26 samples, 2 were both MGIT
and NRA negative. The other 24 were identified
as M. tuberculosis complex by both PNB test and
PRA-hsp65. One NRA result for capreomycin was
indeterminate and one isoniazid well was found
contaminated. As for the remaining results, only one
discordance was observed between both methods.
ThesensitivityandthespecificityoftheD-NRAwas
100% for all drugs except for capreomycin with a
sensitivity of 100% and a specificity of 94.7%. The
detection rate on day 14 was 50% for D-NRA and 9.5%
for MGIT. Mean time to results was 16.5 and 21.9 days,
respectively (P: <0.001). The local cost of supplies
per sample was uS$24.7 for D-NRA and uS$50.5 for
MGIT.
Conclusion: Advantages of this fast colorimetric plate
version include simplification of media dispensing
and/inoculation procedures, and reduction of storage/
84
incubation space. Plate-NRA is simple and appears
to be accurate to detect XDR-TB. In comparison with
the MGIT system, D-NRA was faster and cheaper.
P95
RPOB POLYMORPHISMS IN MyCOBaCTERIUM
TUBERCULOSIS COMPLEx FROM A
POPULATION IN GUINEA-BISSAU
AF Sutre1, A Sanca2, A Mané2, V Henriques2, C
Portugal3, Luisa Sancho3, A Cardoso1, E Paixão1,
CqF Leite4, JI Salem5, A Antunes6, EL Duarte7, S
David1
1
Instituto nacional de Saúde Dr. Ricardo Jorge
(Insa,Ip), Lisbon, portugal
2
Cumura hospital, Cumura, the Republic of
guinea-Bissau
3
Serviço de patologia Clínica, hospital Fernando
Fonseca, amadora, portugal
4
Faculty of pharmaceutical Science – UnESp,
araraquara (Sp), Brazil
5
Instituto nacional de pesquisas da amazônia,
Manaus, Brazil
6
Instituto de higiene e Medicina Tropical/ UnL,
Lisbon, portugal
7
Escola de Ciências e Tecnologia/ ICaaM,
Universidade de Évora, Évora, portugal
The global tuberculosis (TB) threat is difficult to
evaluate in countries with scarce laboratory facilities
where little is known about the genetic basis of drug
resistance, including the rpoB RRDR (rifampicin
resistance determining region) of the Mycobacterium
tuberculosis complex (MTC). The present study
aimed at performing a preliminary evaluation of rpoB
polymorphisms in a DNA set of MTC isolates from
Guinea-Bissau patients.
Ninety-four sputum specimens (74 bleach processed
and 20 unprocessed) were sent to Lisbon for molecular
analysis (n=94) and drug susceptibility testing (n=20).
A 369bp region of the rpoB gene (including the
81bp RRDR), was amplified. Sequencing was used
as the gold standard for the identification of point
mutations.
Two polymorphisms were identified: The alteration
S531L, present in 3.2% of the specimens, and a
new mutation, present in 28.7%, corresponding to
nucleotide and amino acid polymorphism C224T
and S469L, respectively, detected upstream from
the RRDR, and not associated with RMP resistance.
An NCBI BLAST revealed M. tuberculosis K85
(M. africanum) as the only strain presenting this
polymorphism.
The circulation of S531L mutated stains, associated
with RMP resistance, points to the urgent need to
further investigate RMP resistance in this African
region. Moreover, the strains presenting the S469L
polymorphism showed no other mutations suggesting
that its role on RMP susceptibility/ resistance or in
providing a genetic background for the occurrence
of RMP resistance associated mutations should be
further investigated. Also, future genotyping studies
could clarify whether this new mutation is a genetic
signature of some M. africanum strains.
Acknowledgements: This work was supported by
the Luso-American Development Foundation (LACR
Award program).
P121
ExPAND-TB: ExPERIENCE OF ESTABLISHING
TB LABORATORIES IN LIMITED RESOURCES
SETTINGS IN EASTERN EUROPE AND CENTRAL
ASIA
Alexei Korobitsyn1 ; K. Kao, C.N. Paramasivan, D.
Orozco
FInD, geneva, Swizerland
Background:In2009,UNITAIDapprovedafiveyear
project (EXPAND-TB) implemented as a collaboration
between uNITAID, WHO, GLI, FIND and the Stop
TB Partnership GDF, to accelerate access to new,
rapid and WHO endorsed diagnostic technologies for
patients at risk of MDRTB in 27 countries, including
8 in Eastern Europe and Central Asia: Azerbaijan,
Belarus, Georgia, Kazakhstan, Kyrgyzstan, Moldova,
Tajikistan and uzbekistan.
Methods: Political commitment for implementation
of the project was obtained by signing of MOus
with each country Ministry of Health. The status of
selected laboratories in each country was assessed
and the necessary infrastructure and biosafety
upgrades implemented in collaboration with the
national laboratory services and local/international
partners. Equipment, reagents and supplies for liquid
culture and DST, line probe assay (LPA), and rapid
speciation, were procured for each laboratory based
on need. Technology transfer was initiated, including
onsite validation of techniques, quality assurance,
capacity building for staff to use the new tools through
training and regular mentoring in collaboration with
the supporting SRLs. National diagnostic algorithms
were revised and endorsed by local authorities to
incorporate the new diagnostic tools; and quality
assured routine testing and diagnosis of TB and
MDRTB was initiated.
Results: A total of 19 laboratories in 8 countries are
targeted for support under the EXPAND-TB project, 13
of these are already providing quality assured routine
diagnostic services. A cumulative 10,245 MDRTB
cases (34% of regional and 8,9% of global target)
have been diagnosed and reported in 7 countries
excluding Kazakhstan, where EXPAND TB supported
routine diagnostic services are still to start. The
supported technologies have been integrated into the
national diagnostic algorithms but continued support
is required to ensure rational use of them. Lack of
sample transportation in countries is limiting access
to the rapid diagnosis of MDRTB just to patients who
are close to diagnostic locations.
Conclusion: In spite of major obstacles for the project
start up, we have successfully supported TB laboratory
operations in the region, contributing to narrowing the
diagnosis gap for MDRTB cases. The establishment of
TB laboratory networks in resource limited settings is
requiring political commitment, collaboration between
various government departments and partners.
The major limitations to rapidly strengthening the
laboratory diagnosis of MDRTB are the need for
costly infrastructure and biosafety upgrades, human
resources scarcity and high turnover, complex custom
clearance for imported goods and a lack of a sample
transportation system.
P131
COMPARATIVE EVALUATION OF TK SLC-L, THE
RAPID LIqUID MYCOBACTERIAL CULTURE
MEDIUM, WITH BACTEC MGIT
Sakarya Univ. Sch. Med. Medical Microbiology,
Adapazarı, Turkey
Tuberculosis has been for many centuries the most
important of the human infections, with its global
prevalence, devastating morbidity and massive
mortality. Culture is the gold standard method for the
diagnosis of tuberculosis. Rapid mycobacterial culture
systems are important tools with high sensitivity, for
early diagnosis of tuberculosis. The present study
was attempted to assess the efficiency of using TK
SLC-L (Salubris Inc.), by comparing it to MGIT (Becton
Dickinson), in primary isolation of mycobacteria,
from clinical samples. Although TK SLC, biphasic
medium, has been previously evaluated in several
studies, this is the first study evaluating TK SLC-L,
the liquid medium. TK Media have the advantage of
being ready-to use. Clinical specimens from a total of
146 clinically suspected cases of tuberculosis were
studied. The samples were split into two equal parts.
One part was decontaminated and concentrated
using classical NaOH-NALC (Kubica) method and
the other by a new kit, Decomics, which concentrates
the samples by absorbent beads, eliminating the
need for centrifugation. Each processed sample was
evaluated by EZN staining and inoculated into TK
SLC-L and MGIT tubes. TK SLC tubes were incubated
in Mycolor TK and MGIT tubes in BACTEC MGIT
960. Each growth, indicated by automated systems,
was confirmed by making a smear and microscopic
evaluation, after EZN staining. Mycobacteria were
isolated from 46 patients in samples processed by
Kubica method and in 39 patients from samples
prepared by Decomics. Mycobacterial growth was
positive in 35 TK SLC-L and in 34 MGIT tubes in
samples prepared by Kubica method and in 33 TK
SLC-L and in 31 MGIT tubes in samples prepared
85
by Decomics. In samples processed with Kubica
method, average time to growth detection was
18.3 days, median being 15.1 days in TK SLC-L
and 13.0 days, median being 7.7 days in MGIT. In
samples processed by Decomics, average time to
growth detection was 13.9 days, with a median of
11.0 days in TK SLC-L and 10.5 days in MGIT, with a
median of 7.7 days. In samples prepared by Kubica
method and Decomics, contamination rates were
1.3% and 6.2% in TK SLC-L and 13.7% and 9.6%
in MGIT, respectively. Although growth detection time
was approximately 3 to 5 days shorter in average
in MGIT, contamination rate was significantly lower
in TK SLC-L. The total time spent for the repetition
of cultures for contaminated samples in MGIT may
make the returning time of culture results equal to the
longer detection time required by TK SLC-L. It can be
concluded that TK Culture System using TK SLC-L is
a good system that may be alternative to other rapid
mycobacterial culture systems.
P138
DEVELOPMENT OF A NEW DNA MICROARRAY
PLATFORM FOR THE DETECTION OF MDR
TUBERCULOSIS
Andrea M. Cabibbe1, Klaus Reither2, Daniela M.
Cirillo1, EDCTP TB CHILD Consortium3, FP7 TM
REST Consortium1
1
Emerging Bacterial Pathogens Unit, Division
Italy
2
Swiss Tropical and Public Health Institute,
Basel, Switzerland
3
Ifakara Health Institute, Bagamoyo, Tanzania
The emergence of multi drug resistant (MDR)
tuberculosis (TB), defined as resistance to at least
rifampin (RIF) and isoniazid (INH), is jeopardizing
TB control in high burden Countries and Eastern
Europe. One of the greatest challenges to the control
of DR is the lack of adequate laboratory facilities to
perform drug susceptibility testing. For this reason
more advanced, fast and affordable technologies
are needed to strengthen laboratory capacity for
diagnosis of DR TB.
Afirstgenerationofanewrapidmultiplexedmolecular
diagnostic assay for detection of MDR TB by a lab-onchip (LoC, vereMTBTM) was developed within the FP7
TM REST Project. Aim of this study is to evaluate the
performances of the improved version of vereMTBTM
assay.
The vereMTBTM provides an all-in-one device for
fast-PCR amplification and detection of targets on
a low-density microarray. Most clinically relevant
mycobacterial species are identified by targeting
the 16S rRNA gene and IS6110 insertion sequence,
whereas DR is detected by analyzing rpoB, katG, and
86
inhA as the most frequently mutated genes involved
in the MDR phenotype in M. tuberculosis complex
(MTBC). A multiplex PCR to amplify all the target
genes was developed (to work in the microfluidic
chamber of the LoC device).
Specific probes for species identification and DRrelated mutation detection were included in the array.
The assay allows identifying MTBC, and 10 clinically
relevant non-tubercular mycobacterial (NTM) species,
including M. avium and M. intracellulare. The assay
detects the following mutations involved in DR TB:
D516v, S531L, H526D/y (rpoB), S315T (katG), and
c-15t, t-8c, t-8a (inhA).Othermutationsareidentified
by a negative signal from wild type probes. A second
generation of the chip improved the performances
of the assay, optimizing the multiplex PCR, and
includingaspecificprobeforL511Pmutation(rpoB),
3 replicates on the array for each probe instead of 2,
andamplificationcontrolsforeachgene.
The overall sensitivity of this new assay on clinical
isolates is >99%, >95%, and >97% for MTBC, NTM,
and MDR detection, respectively, while specificity is
>98%, >98%, and >97%, respectively. A preliminary
evaluation on clinical specimens showed valid results
in the > 94% of cases on smear positive cases.
This integrated PCR and microarray chip tool
represents an innovation for its simplicity of use,
rapidity and cost-effectiveness, and it’s particularly
suitable for different diagnostics purposes, making it
indicated for the laboratory routine.
P140
EVALUATION OF PHENOTYPIC AND GENOTYPIC
DRUG SUSCEPTIBILITY TESTING METHODS
FOR FLUOROqUINOLONE RESISTANCE IN M.
TUBERCULOSIS
Nele Coeck1, Bouke de Jong2, Leen Rigouts1
1
Institute of Tropical Medicine, Department of
Biomedical Science, Mycobacteriology unit,
Antwerp, Belgium; University of Antwerp,
Faculty of Pharmaceutical, Biomedical and
Veterinary Sciences, Antwerp, Belgium
2
Institute of Tropical Medicine, Department of
Biomedical Science, Mycobacteriology unit,
Antwerp, Belgium
Drug-resistant tuberculosis increasingly hinders the
success of TB control programs. An important part
(35-85%) of clinical M. tuberculosis isolates with
phenotypic fluoroquinolone (FQ) resistance have
mutations in the gyrA gene whereas gyrB mutations
are seen occasionally. Clinical data show that Fqs are
crucial for successful outcome of MDR-TB treatment.
However, the extent of in vitro cross-resistance to the
various generations of Fqs remains unclear, including
the relation of minimal inhibitory concentrations (MICs)
for these different Fqs to gyrAgyrB mutations.
MIC determination of ofloxacin (OFX), levofloxacin
(LVX), moxifloxacin (MFX) and gatifloxacin (GFX)
was done in Löwenstein-Jensen (LJ) - as the gold
standard - and the colorimetric resazurin microtiter
assay (REMA) in 54 M. tuberculosis isolates which
had been identified as OFX-resistant in routine
diagnostic testing (7H11 proportion method, cut-off 2
µg/mL). Thirty-six isolates were simultaneously tested
for OFX resistance in MGIT960 (proportion method).
In all media, we used 2 µg/mL as cut-off for OFX, 1
µg/mL for LvX, and 0,5 µg/mL for GFX and MFX.
Although cross-resistance between the various Fqs
in all OFX-resistant isolates was noticed, MICs of
the newer generation Fqs (MFX and GFX) were
systematically lower. Approximately one third of the
OFX-resistant isolates (40,54%) had no gyrAgyrB
mutations, of which 66,67% had MICs for OFX of
8 µg/mL or higher in LJ. When comparing OFX
susceptibility in REMA and MGIT towards the gold
standard, 13 isolates were tested OFX-resistant in
both LJ, REMA and MGIT, 7 isolates (6 wild-type and
1 with gyrA mutations) were susceptible in both REMA
and MGIT (but resistant in LJ), 8 isolates (3 WT and 5
with gyrA mutations) were susceptible in REMA (but
resistant in LJ and MGIT) and 1 isolate (WT) was
resistant in MGIT (but susceptible in LJ and REMA).
Seven strains were found to be OFX-susceptible in all
3 testing methods. Of the 18 strains which were tested
only in REMA and LJ, 4 were susceptible in REMA,
yet resistant in LJ (1 WT and 3 with gyrA mutations),
and 9 were susceptible in both REMA and LJ.
These preliminary results show cross-resistance
between the various Fqs, although new generations
of Fqs had systematically lower MICs. In addition,
probable false-susceptible results in REMA suggest
the need for lowering the current used critical
concentrations of Fqs in liquid medium (e.g. REMA
and MGIT), however a more extensive validation
experiment is warranted. Furthermore, the high
proportion of wild-type gyrAgyB yet phenotypic Fqresistance suggests the involvement of alternative
resistance mechanisms, such as efflux pumps.
Hence, an in-depth analysis to these mechanisms is
currently ongoing.
P141
TB PAN NET CONSORTIUM: SHARED
DATABASES FOR THE CHARACTERIZATION OF
MUTATIONS INVOLVED IN DRUG-RESISTANT M.
TUBERCULOSIS PHENOTYPE
Andrea M. Cabibbe1, Paolo Miotto1, Emanuele
Borroni1, Ilaria C. Valente1, Silke Feuerriegel2,
Petras Stakenas3, Ewa Augustynowicz-Kopeć4,
Vanessa Mathys5, Sven Hoffner6, Stefan
Niemann2, Daniela M. Cirillo1
1
Italy
Molecular Mycobacteriology group,
national Reference Center for Mycobacteria,
Forschungszentrum Borstel, Borstel, germany
3
Department of Immunology and Cell Biology,
Institute of Biotechnology, vilnius University,
vilnius, Lithuania
4
Microbiology Department, national Tuberculosis
and Lung Diseases Research Institute, Warsaw,
poland
5
Tuberculose & Mycobacteries, Scientific
Institute of public health – Institut pasteur,
Brussels, Belgium
6
Department for preparedness, Swedish Institute
for Communicable Disease Control, Solna,
Sweden
2
The FP7 TB PAN NET Project aims at providing
improved tools to fight drug resistant tuberculosis
(DR TB), and assisting industry in the development
of new diagnostics and treatment regimens. Better
understanding of the relationship relying between the
genetic markers of DR and the clinical outcome will
permit the establishment of new diagnostic tools with
improved effectiveness.
We created large databases to collect data on
the genetic variants observed in susceptible and
resistant clinical isolates. Mutations occurring in
genes encoding putative targets for anti-TB drugs
detected by sequencing have been included together
with phenotypic and genotyping data to evaluate the
correlation between the polymorphisms detected, the
DR phenotype observed and the genetic background.
Minimum Inhibitory Concentration and enzymatic
activitywereconsideredtosolvespecificphenotypegenotype correlations.
In particular, we focused on pyrazinamide (PZA,
about 1200 isolates), fluoroquinolones (FQ) and
second-line injectable drugs (SLID, 412 isolates). We
included data on specimens leading to uncommon
pattern on molecular assays approved by the World
Health Organization for fast detection of rifampin
(RIF)-R (185 isolates), and on ethionamide (ETH, 52
isolates). All samples were typed by spoligotyping; to
solve high clustering rate associated with the Beijing
lineage, MIRu-vNTR was included.
Concerning the pncA gene leading to PZA-R, we
detected that the following codons account for 6570% of mutations: 6 to 15, 46 to 70, 75 to 85, 131
to 145, 171 to 175, and nucleotides -11, -7 in the
promoter region. Nearly 90% of SLID-R cases was
detected by targeting rrs, eis, and gidB; in addition,
certain variations, especially in gidB, appear to be
phylogenetically informative rather than markers for
DR. The 87% of Fq-R cases harbored mutations in
gyrA and gyrB. The S512T, M515I, H526N, S531W,
L511q, D516T and other substitutions or deletions
accounted for about 50% of “uncommon” patterns in
RIF-R cases; analysis of the rpoA and rpoC genes
was also included for monitoring compensatory
mutations. The 26.9% of ETH-R strains harbored
mutations scattered along the entire ethA gene.
Our study provides a first insight on the European
87
SNPs-genotype distribution in resistant clinical
isolates. Large public data sets constitute the basis for
new understanding of molecular patterns associated
with DR mechanisms and are precious tools for the
design of novel molecular assays.
P142
DYNAMIC MICROCOLONY GROWTH
MONITORING FOR DRUG SUSCEPTIBILITY
TESTING OF ETHAMBUTOL AND
PYRAZINAMIDE
Alice den Hertog1, Sandra Menting1, Ernst
Smienk1, Sarah Sengstake1, Sven Hoffner2,
Richard Anthony1
1
KIT Biomedical Research, amsterdam,
netherlands
2
A complete overview of the susceptibility to available
drugs is crucial for the provision of effective therapy
and interruption of transmission of drug resistant
pathogens. unfortunately, drug susceptibility testing
(DST) of some antimycobacterial drugs is technically
challenging.
We developed a novel culture system for DST, in
which the growth of microcolonies from individual
CFus is monitored and microcolonies can be
(transiently) exposed to drugs to measure growth rate
and susceptibility of individual colonies. We previously
demonstrated we could distinguish rifampicin (RIF)
susceptible and resistant strains of Mycobacterium
tuberculosis within 8 days after inoculation and after
only 24 hours exposure to RIF (den Hertog PLoSONE
2010).
Whereas rifampicin DST is relatively uncomplicated,
DST is more difficult to perform reproducibly for
ethambutol (EMB), a drug for which the MIC values of
resistant are close to those of susceptible strains, and
for pyrazinamide (PZA) which is active only at low pH
which can also inhibit mycobacterial growth.
A panel of 20 tuberculosis strains with known
ethambutol susceptibility was tested blindly and
classified based on comparison with 4 strains with
known susceptibility/resistance. This yielded a 95%
accurateclassification(19/20strains).Onlyonestrain
could not be classified due to a contamination, but
whenretested,thisstrainwasalsocorrectlyclassified,
yielding a 100% accuracy.
Currently we are exploring the possibility for performing
DST for PZA by transiently exposing microcolonies
onacidifiedmediumwithPZA,andmeasuringpostexposure growth rate in standard medium.
P144
PNCA GENE MUTATIONS AND PYRAZINAMIDE
RESISTANCE IN SWEDISH MULTIDRUGRESISTANT TUBERCULOSIS 2003-2013
Mikael Mansjö, Jim Werngren, Sven Hoffner, Ylva
Lidén
Background:Pyrazinamide(PZA)isafirstlinekey
drug in the treatment of tuberculosis (TB), including
multidrug-resistant (MDR) TB susceptible to PZA.
Inside the bacteria, the prodrug PZA is activated by
the bacterial enzyme pyrazinamidase (PZase) which
converts PZA into pyrazinoic acid. PZase is encoded by
the pncA gene and the correlation between phenotypic
PZA resistance and the presence of mutations in the
pncA gene has been well established.
Objective: By comparing sequencing of the pncA
gene and data from the Bactec MGIT 960, we
determined the prevalence of PZA resistance among
clinical Swedish MDR-TB strains isolated in Sweden
between 2003 and 2013.
Method: 119 clinical TB isolates defined as MDR
were tested for PZA resistance in the Bactec MGIT
960 system and subsequently screened for mutations
within the pncA gene.
Results: Preliminary results indicate that 57% of the
MDR strains are PZA resistant. In addition, 94% of the
PZA resistant strains have a mutation in the pncA gene
or its putative promoter. Of the PZA susceptible strains,
77% have a wild type pncA gene. The silent mutation
Ser65Ser (C195T; TCC65TCT) was detected in 14%
of the susceptible samples. Moreover, the sensitivity
andspecificityofpncA sequencing, using the Bactec
MGIT 960 system as gold standard, was determined
to be 94% and 90%, respectively. 18 out of 37 clinical
MDRisolates(49%)wereclassifiedasPZAresistant
duringthefirstfiveyear-periodwhereas50outof82
clinical MDR isolates (61%) were classified as PZA
resistant during the latter five year-period (including
the three isolates from 2013).
Conclusion: The results from the last ten year period
demonstrate a slight increase of PZA resistance
among Swedish MDR cases. Additionally, the
detection of pncA gene mutations, or their absence,
wasconfirmedasausefulmethodforpredictingPZA
resistance.
P160
THE INFLUENCE OF GLUTOxIM ON THE
RESISTANCE OF MyCOBaCTERIUM
TUBERCULOSIS (MTB) STRAINS TO ISONIAZID
Olga Manicheva1, Natalya Solovyeva1, Viacheslav
Zhuravlev1, Victor Antonov2, Dmitriy Aizikov3,
88
Marina Shulgina1
1
Saint-petersburg State Research Institute of
phthisiopulmonology, St. petersburg, Russia
2
Military Medical academy, St. petersburg,
Russia
3
petrozavodsk State University, petrozavodsk,
Russia
Background: Isoniazid is the most effective drug
used for the treatment of tuberculosis. Resistance to
isoniazid and rifampicin (MDR) is prevalent in Russia
and a serious threat to the battle against tuberculosis.
Therefore, research on drugs which make it possible to
reduce the isoniazid minimum inhibitory concentration
(MIC) for drug-resistant mycobacterium strains is
of capital importance. Preliminary studies revealed
that Glutoxim (a commercial immunostimulator) fully
converts isoniazid into its active form (isonicotinic
acid) in the presence of hydrogen peroxide. Glutoxim’s
ability to enhance the effect of isoniazid on resistant
mutated mycobacterium strains was studied.
Methods: Isoniazid-resistant Mtb clinical isolates were
studied. Indirect testing for phenotypic drug-resistance
was performed using the absolute concentration
method on Löwenstein-Jensen media. A TB-Biochip
microarray system was used to detect isoniazid
resistance mutations. The following procedure was
used to determine MICs of various isoniazid/Glutoxim
combinations on H37Rv and three isoniazid-resistant
(due to mutations of the katG, ahpC and inhA genes)
Mtb isolates: a modified checkerboard method on
OADC-enriched Middlebrook 7H9 Broth, addition of
Thiazolyl Blue and spectrophotometric (at 630 nm)
evaluation of growth.
Results: Glutoxim alone had no effect on the growth
of any of the tested Mtb strains. The initial isoniazid
MIC for H37Rv was 0.062 μg ⁄ ml; Glutoxim (503.1μg⁄ml)loweredthisto0.031μg⁄ml.Theinitial
isoniazid MIC for the katG-mutated (Ser315 →Thr)
strainwas3.5μg⁄ml;Glutoximloweredthisbyafactor
of1.6μg⁄ml.Theinitialisoniazid MICforthekatG
(Ser315→Thr1,IIe335→Val)+ahpC-T10mutated
strain was 20 μg ⁄ ml; Glutoxim lowered this by a
factor of two. The inhA-T15 mutant’s initial isoniazid
resistance was found to be inferior (0.62 μg ⁄ ml);
Glutoximloweredthisevenfurtherto0.31μg⁄ml.
Conclusions: Glutoxim lowered the isoniazid MIC
by a factor of 1.6-2 in sensitive Mtb H37Rv, and also
in clinical isolates with strong/weak drug-resistance
caused by various genetic mutations (katG, ahpC,
inhA).
P173
GENETIC CHARACTERIZATION OF
PYRAZINAMIDE-RESISTANT M. TUBERCULOSIS
COMPLEx ISOLATES IN AN ITALIAN NORTHEASTERN AREA DURING A FOUR YEAR PERIOD
Mario Rassu2, Michela Pascarella2, Riccardo
Manganelli1, Giorgio Palù1
1
Department of Molecular Medicine, University of
padua, padua, Italy
2
Department of Microbiology, S. Bortolo hospital,
vicenza, Italy
Pyrazinamide (PZA) is an important first-line
antitubercular drug that plays an unique role in
achieving shortened chemotherapy in combination
with isoniazid, rifampicin and ethambutol.
We examined 52 PZA-resistant clinical strains isolated
in the Laboratories of Microbiology and virology of the
Hospitals of Padua and vicenza (Jan. 2009 – Dec.
2012).
7 strains were collected during 2009,13 during 2010,
23 during 2011 and 9 during 2012.
The aim of this study was to evaluate the role of pncA
gene mutations as a marker for the detection of PZA
resistance in M. tuberculosis (Mtb).
PZA susceptibility testing (PZA-ST) was performed
usingtheMGIT960system(M960)withanacidified
culture medium (pH 5.9). Fully susceptible M.
tuberculosis strain H37Rv (ATCC 27294) was included
as reference strain.
12 out of 52 PZA-resistant isolates were multidrugresistant TB strains, 4/52 had varied drug susceptibility
patterns and 36/52 were susceptible to the others
first-linedrugs.
Mutations in the pncA gene were detected in 27 out
of 52 MGIT960 PZA-resistant isolates. The majority
of these (23/27) had different unique point mutations
resulting in nucleotide substitutions. As expected, all
15 M. bovis strains had a His57asp mutation. One
strain had an 8 bp deletion, between nt 115 and 122:
at the best of our knowledge this deletion was not
identifiedinItalyyet.
Three strains had mutations in the upstream regulatory
region: one of them had a nucleotide change located
at -11 nt, one at - 12 nt and one strain had a 35 bp
deletion between -10 and -44. The last two mutations,
also,arenotidentifiedinItalyyet.
PZA-susceptibility tests of 25 PZA-resistant isolates
that had no mutations in the pncA gene or in their
promoter (WT) were repeated using a reduced
inoculum (RI) to evaluate major errors recently
reported in literature.
In the RI assay 10 samples turned out to be susceptible
and24wereconfirmedtoberesistantsuggestingthe
existence of an alternative mechanism of resistance.
It remains to be determined if the strains which did
notconfirmtheirresistanceafterRIhavelowlevels
of resistance.
The analysis of the pncA gene provides rapid and
useful information regarding PZA susceptibility in Mtb
and may therefore contribute to early optimization of
treatment. Furthemore, pncA sequencing may be a
useful support for the phenotypic PZA–ST.
Marta Peracchi1, Loredana Fallico1, Marta Viero1,
89
P174
P179
INTEGRATING THE xPERT MTB/RIF ASSAY
IN A DIAGNOSTIC WORK FLOW FOR
RAPID DETECTION OF MyCOBaCTERIUM
TUBERCULOSIS IN A LOW PREVALENCE AREA
THE EFFICIENCY OF A NEW DECONTAMINATION
AND CONCENTRATION KIT WHICH ELIMINATES
CENTRIFUGATION, IN ISOLATION OF
MYCOBACTERIA FROM SPUTUM SAMPLES
Akos Somoskovi1, Claudia Ritter1, Vanessa
Deggim1, Antje Voit2, Eric C. Böttger1, Guido V.
Bloemberg2
1
Institut für Medizinische Mikrobiologie
Universität Zürich, Zürich, Switzerland;
nationales zentrum für Mykobakterien,
Universität zürich, 8006 zürich, Switzerland
2
Institut für Medizinische Mikrobiologie
Sakarya Un. Sch Med. Medical Microbiology,
Turkey
The Xpert MTB/RIF assay is a rapid and fully
automated real-time PCR assay. Its high cost and
limited sensitivity in comparison with other PCR MTB
test systems preclude the Xpert MTB/RIF assay as
a diagnostic procedure for routine direct detection of
MTB in low prevalence areas.
The aim of this study was to integrate the Xpert MTB/
RIF assay into a diagnostic work flow for urgent
clinical specimens and to evaluate the performance
of the Xpert MTB/RIF assay as a primary screening
test for urgent clinical specimens with the view to
replace insensitive and laborious microscopy with
low discriminatory power. During a two-year period
79 specimens submitted for emergency testing were
analyzed with the Xpert MTB/RIF assay.
Our results provide strong evidence that for respiratory
samples in a low prevalence setting the Xpert MTB/
RIF assay improves accurate primary screening and
is well suited to replace smear microscopy for urgent
specimens. The high costs of the Xpert MTB/RIF
assay could be compensated by its fast and walkaway
methodology compared to the laborious procedure of
AFB microscopy which involves tedious reading of
slides requiring well experienced personnel for quality
results.
As described earlier we have identified false RMP
resistance by the Xpert MTB/RIF assay in two
respiratoryspecimens.Theseresultshaveasignificant
impact on accurate screening for RMP resistance in
a low tuberculosis and MDR-TB prevalence setting
such as Switzerland. More recently, we have also
detected false negative RMP resistance results by the
Xpert MTB/RIF assay, which were associated with a
rpoB Leu533Pro mutation as shown by direct nucleic
acid sequencing.
As a consequence we strongly recommend that in
low prevalence settings RMP resistant results by the
Xpert MTB/RIF assay should always be confirmed
by an alternative molecular assay until conventional
DST is available.
90
Two decontamination and concentration kits,
Mycoprosafe and Decomics (Salubris Inc.)
were compared for mycobacterial isolation rate,
contamination rate and time to mycobacterial detection
in rapid culture systems. Mycoprosafe contains
all materials needed for application of classical
NaOH-NALC decontamination and concentration
(Kubica) method. Decomics is a new kit which
removes decontamination and neutralization fluids
by absorbent beads and thus eliminates the need for
centrifugation. A total of 146 sputum samples, were
collected from tuberculosis suspected patients. The
samples were split into two equal parts and processed
by Mycoprosafe and Decomics. Processed samples
were examined by AFB staining and were inoculated
into TK SLC-L and MGIT culture tubes and incubated
in Mycolor TK and BACTEC MGIT 960 instruments,
respectively. Any growth was confirmed by AFB
staining. Contamination control for MGIT was done
by subculturing to sheep blood agar; contamination
was followed in Mycolor TK which can predict
contamination, as well as growth. Smear positivity
for AFB was 11.6% by both methods. From samples
processed by Mycoprosafe, mycobacteria were
isolated in 35 (24.0%) TK SLC-L, in 34 (23.3%) MGIT
tubes and 46 (31.7%) all together in any type of media.
From samples processed by Decomics mycobacteria
were isolated in 33 (22.6%) TK SLC-L, in 31 (21.2%)
MGIT tubes and 39 (26.7%) all together in any type
of media. In samples processed with Mycoprosafe,
average time to growth detection was 18.3 days in TK
SLC-L and 13.0 days in MGIT. In samples processed
by Decomics, average time to growth detection was
13.9 days in TK SLC-L and 10.5 days in MGIT. In
samples prepared by Mycoprosafe contamination
rates were 1.3% in TK SLC-L and 13.7% in MGIT.
With Decomics, contamination rates were 6.2% in TK
SLC-L and 9.6% in MGIT. In summary, slightly lower
isolation rates were obtained in samples processed by
Decomics which may be due to higher contamination
rates, since mycobacteria were isolated from 6 samples
processed by Mycoprosafe which were contaminated
by Decomics. Contamination rate in Decomics can
be lowered by increasing decontamination time from
10 to 15 minutes as recommended in the application
procedure. Decomics speeds up mycobacterial
detection time 3 to 5 days depending on the rapid
culture method used. Decomics, which lowers sample
processing time from 45 to 25 minutes and eliminates
the need for centrifugation, may be a good alternative to
classical Kubica method in laboratories with elaborate
capabilities and may enable to do decontamination
and concentration and thus mycobacterial culture,
the laboratories where equipment and methods like
centrifugation are limited.
P180
RAPID DIAGNOSIS OF MULTI- AND
ExTENSIVELY DRUG RESISTANT
TUBERCULOSIS AMONG PATIENTS WITH HIGH
RISK OF TB RESISTANCE
Valeriu Crudu, Elena Romancenco, Ecaterina
Noroc, Nadejda Turcan, Galina Blagoadeteleva
Center for Health Policies and Studies;
phthisiopneumology Institute, Chisinau,
Republic of Moldova
Background: Rapid diagnosis of multi- and
extensively drug resistant tuberculosis (MDR&XDR
TB) is essential for the prompt initiation of effective
second line therapy to improve treatment outcome
and limit transmission of TB resistance. The
GenoType MTBDRplus 2.0 and GenoType MTBDRsl
assay are a commercially available line-probe
assay that rapidly detects M.tuberculosis complex,
as well as the most common mutations associated
with Rifampicin, Isoniazid, Fluoroquinolopne,
Aminoglycoside
and
Ethambutol
resistance.
Objectives of study were to evaluate the GenoType
MTBDRplus 2.0 and MTBDRsl assay for rapid
diagnosis of MDR&XDRTB among new and
retreatment cases, with high risk of TB resistance.
Method: A total of 1405 patients with results of
MTBDRplus 2.0 and 194 patients with results of
MTBDRsl were analyzed. From these, the fourth part
of specimens was performed directly from smearnegative (33,5%) and smear-positive (66,5%) sputum.
The strains used for molecular testing were isolated
byliquidmedia(MGIT960)andDSTforfirstlinedrugs
were performed and compared with LPA results.
Results: The RIF resistance was detected in total
in 785 (55,9%) cases and INH was resistant in
939 (66,8%). The MDR TB were detected in 646
(46,0%) cases. The most common mutations for
RIF resistance was rpoB MuT3 (86,8%) and for INH
resistance katG MuT1 (97,7%). All the patients with
MDRTB were investigated for second line TB drug
resistance. The results of MTBDRsl: 60 (30,9%)
of patients were resistance to Fq, resistance to
AG were detect in 26 (13,4%) cases and EMB
resistance were detected in 44 (22,7%) cases. The
most common mutations for Fq were gyrA MuT1
and gyrA MuTC (36.7% and 41,7%), for AG - rrs
MuT1 (73,1%) and for EMB - embB MuT1B (75.0%).
Conclusion: Effective control of drug resistant
tuberculosis requires massive scaling-up of culture
and DST capacity, and simultaneous use of rapid
molecular assays. The MTBDRplus ver.2 and
MTBDRsl assay are sensitive and specific tools for
diagnosis of MDR&XDRTB in sputum specimens
and culture strains. The short turnaround times and
the potential for rapid screening of large numbers of
specimens make it suitable as a first-line screening
assay for TB drug resistance. With effective planning
and logistics, the molecular based methodologies can
be successfully introduced into a reference laboratory
setting in high burden MDRTB country. High rates of
MDR&XDRTBmaketheintroductionofsuchassays
particularly useful.
P181
EVALUATION OF SECOND-LINE
ANTITUBERCULOSIS DRUGS SUSCEPTIBILITY
TESTING OF MULTIDRUG-RESISTANT
(MDR) ISOLATES OF MyCOBaCTERIUM
TUBERCULOSIS USING ETEST
Hulya Simsek1, Gülnur Tarhan2, Salih Cesur3
1
national public health agency, ankara, Turkey
2
Ahi Evran University, Kırşehir, Turkey
3
Etlik Training and Research hospital, ankara,
Turkey
Background: Multidrug resistant tuberculosis
(TB) is a major osbstacle for effective treatment of
tuberculosis worldwide. Rapid detection of resistance
allows appropriate intervention to control the disease.
Etest (AB BIODISK, Solna, Sweden) is a simple
technique that provides quantitative drug susceptibility
results for M. tuberculosis in 5 to 10 days from a
culture grown at low cost. The aim of this study was
to determine the effectiveness of the Etest to detect
second-line (Kanamycin, Ofloxacin, Ethionamide,
Linezolide ) antituberculosis drug susceptibility of
multi drug-resistant tuberculosis.
Methods: A total of 122 multidrug resistant M.
tuberculosis isolates were tested. First line and
second line antimicrobial susceptibility testing was
performed by proportion method using LowensteinJensen medium (PMLJ). The antibacterial activities of
kanamycin,ofloxacin,ethionamide,linezolideon100
clinical isolates of Mycobacterium tuberculosis were
determined Etest.
Results: Results were obtained in 6 to 10 days by
Etest. When 119 of 122 M. tuberculosis isolates
were susceptible to four second line drugs, three
were resistant to ethionamide and kanamycin. The
correlation between E test and standard proportion
dilutionmethodwas98%kanamycin,96%ofloxacin,
98% ethionamide, 100% linezolide
Conclusion: The Etest method is suitable for testing
the agents evaluated against M. tuberculosis. This
study supports the utility of Etest for timely detection
of drug resistance in M. tuberculosis and for use in
tuberculosis control programs.
91
P182
EVALUATION OF THE PRESENCE OF
MYCOBACTERIA BELONGING TO THE
MyCOBaCTERIUM TUBERCULOSIS COMPLEx
IN ANIMAL TISSUES BY REAL-TIME PCR
Pedro Costa1, Ana Ferreira2, Ana Amaro3, Isabel
Couto4, Miguel Viveiros5, João Inácio3
1
Unidade de produção E Saúde animal,
Instituto nacional de Investigação agrária
E Veterinária, Lisbon, Portugal; Grupo de
Micobactérias, Unidade de Ensino e Investigação
de Microbiologia Médica, Instituto de higiene e
(IHMT/UNL), Lisbon, Portugal;
2
Unidade de produção E Saúde animal, Instituto
nacional de Investigação agrária E veterinária,
Lisbon, Portugal; Instituto de Ciências
Biomédicas de abel Salazar, Universidade do
porto, porto, portugal
3
Unidade de produção E Saúde animal, Instituto
nacional de Investigação agrária E veterinária,
Lisbon, portugal
4
grupo de Micobactérias, Unidade de Ensino e
Investigação de Microbiologia Médica, Instituto
de higiene e Medicina Tropical, Universidade
Nova de Lisboa (IHMT/UNL), Lisboa, Portugal;
Centro de Recursos Microbiológicos (CREM),
Universidade nova de Lisboa, Lisbon, portugal
5
grupo de Micobactérias, Unidade de Ensino e
Investigação de Microbiologia Médica, Instituto
de higiene e Medicina Tropical, Universidade
nova de Lisboa (IhMT/UnL), Lisbon, portugal
The Mycobacterium tuberculosis complex (MTC)
includes several closely related pathogenic species,
which includes M. tuberculosis, the principal agent of
human tuberculosis, M. bovis and M. caprae, agents
of bovine and caprine tuberculosis, respectively.
Bovine tuberculosis is nowadays subjected to
costly eradication programs in most European
union countries, involving the laboratorial testing of
samples from suspected animals for the definitive
confirmationofthepresenceofMTC.Thedetection
of MTC members in biological samples from livestock
and other animals is mainly based in lengthy and
cumbersome conventional methods involving
histological analysis and culture of the agents. In the
present work we have developed a novel and simple
IS6110-targeted taqman-based semi-nested realtime PCR assay yielding extremely high sensitivity
andspecificityforthedirectdetectionofMTCspecies
in animal tissues. Spiked tissue samples and one
hundred and eighteen lymph nodes and lung tissue
samples from slaughtered animals suspected of
having tuberculosis were used for optimization and
evaluation of the assay. Tissues were homogenized
andDNAextractedbyacommercialkit.Amplification
assays enabled the detection of MTC mycobacteria
directly in tissue samples with high sensitivity (98%)
92
and specificity (100%), using a combination of
histological and bacteriological approaches as goldstandard. Additionally, several tissue samples negative
for the isolation of MTC agents, but showing lesions
compatible with tuberculosis, were found to yield
positiveamplificationresultsforMTCwiththeseminestedreal-timePCRapproach.Theco-amplification
of the bovine β-actin gene ruled out the inhibition
of the PCR reactions by inhibitory components
of tissues or excess of bovine DNA. The IS6110targeted semi-nested real time PCR optimized in this
work represents a significant advance for the rapid
and accurate detection of MTC members directly in
animal tissue samples, capable of being introduced in
the routine diagnostics of veterinary laboratories.
P193
EVALUATION OF THE xPERT MTB/RIF ASSAY
FOR THE DIAGNOSIS OF TUBERCULOSIS
Ali Albay, Ozgul Kisa, Mustafa Guney, Gokselin
Dogan, Kemal Tekin
Department of Medical Microbiology, gulhane
Military Medical academy and School of
Medicine, ankara, Turkey
Objectives: The resurgence of tuberculosis and
emergence of multidrug-resistant strains of M.
tuberculosis have stimulated the development of new
diagnostic methods. We have conducted a study to
evaluate an automated tuberculosis assay (Xpert
MTB/RIF) for the presence of M. tuberculosis and
resistance to rifampin.
Methods: In our study we compared Xpert MTB/RIF
assay by the culture methods. The cultures of 389
various routine clinical samples were performed by
BACTEC MGIT 960 culture system (BD, uSA) and
solid Löwenstein-Jensen (LJ) culture media. And also
we tested these clinical samples with Xpert MTB/
RIF system. 171 of our samples were pulmonary
specimens and 218 were extrapulmonary samples.
Results: We detected 23 samples as positive by
Xpert MTB/RIF system and 22 of patient samples
were positive by and Löwenstein-Jensen (LJ) culture
media. 366 samples were found as negative for M.
tuberculosis by Xpert MTB/RIF system and 367 were
negative by culture methods. Fourteen of the culture
positive samples were smear negative. 13 of these
smear negatives were determined as positive for M.
tuberculosis by Xpert MTB/RIF system. Two of the
M. tuberculosis strains were detected as resistant to
rifampin at the beginning by Xpert MTB/RIF system.
This was confirmed by BACTEC MGIT 960 culture
system later on. There was a discordance between
Xpert MTB/RIF system and BACTEC MGIT 960
culture system for one of rifampin resistant strains.
It was resistant to rifampin by BACTEC MGIT 960
culture system but sensitive by Xpert MTB/RIF
system. According to culture results, the sensitivity of
Xpert MTB/RIF system for pulmonary specimens was
100% (16/16) for both smear positive and negative
specimens. For extrapulmonary specimens the
sensitivity and specificity of the system were 83.3%
(5/6) and 99.5% respectively.
Conclusion: Rapid laboratory detection and
identificationofM. tuberculosis is very important for
the treatment of tuberculosis. And also, detection of
resistance to rifampin is of particular importance, as
it is a marker for multidrug-resistant M. tuberculosis
strains. So, Xpert MTB/RIF is a novel automated
molecular diagnostic system for the early diagnosis
of tuberculosis recently endorsed by the World Health
Organization.
P199
RAPID DETECTION OF M. TUBERCULOSIS
DRUG RESISTANCE TO SECOND LINE
ANTIBIOTICS IN SPUTUM SAMPLES BY USING
THE MULTI-COMPETITIVE ALLELE-SPECIFIC
REAL-TIME PCR
Yulia Alyapkina1, Michail Vladimyrsky2, Marina
Lapenkova1, Dmitry Varlamov1
1
Jsc “Syntol”, Moscow, Russia
2
Research phthisiopulmonology Institute of
Sechenovs Moscow University, Moscow, Russia
The rapid spread of Extensive Second Line Drug
Resistant (XDR-TB) tuberculosis and wider use of
fluoroquinolones and other second line antibiotics
requires development and introduction of new
rapid methods for detection of resistance to these
medications. Recently we designed original multicompetitiveallele-specificreal-timePCRmethodfor
detection of main mutations in the rpoB, katG, inhA,
embBgenesassociatedwithfirstlineantibioticsdrug
resistance. Method is based on using simultaneously
a set of 5’-fluorescent allele-specific primers and
linear fluorogenic DNA probe detecting MTB. The
assay allows detection up to three independent point
mutations in one tube. If a sample does not include
mutations the standard real-time PCR will take place
and only the fluorescence of the fluorogenic DNA
probe will increase. If a sample contains mutated
DNA then the 5’-fluorophore labeled allele-specific
primer will release the quencher to take part in the
reactionandthefluorescenceincrementofboththe
fluorogenicDNAprobeandtheallele-specificprimer
will be registered. The technology was employed
for detection of gyrA and rrs genes DNA mutations
associatedwithdrugresistancetofluoroquinolones,
amykacin and capreomycin. 132 sputum samples
from previously treated patients with unknown
resistance were studied. To detect the corresponding
mutations, quantitative real-time PCR was performed,
and the number of MTB cells was determined by
analysis IS6110 and regX3 DNA gene copies. If no
less than 102 MTB cells were present, mutational
analysis with respect to rpoB, katG, inha, gyrA and
rrs genes mutations associated with drug resistance
was conducted. For this purpose, DNA fragments
from the relevant genes were amplified using a
preliminary short (20 cycles) round of multiplex
PCR. After that, mutations were analyzed by a set
of specially constructed fluorescent marked allelespecificprimersasdescribedformerly.For68samples
with more than 102 MTB cells mutational analysis
was conducted. Results of fist line antibiotics drugresistance analysis: 51 (75%) samples were MDR, 7
(10,3%) – monoINH-resistant, 2 (2,9%) – monoRMPresistant, 8 (11,8%) – susceptible. Results of second
line antibiotics drug-resistance analysis (from 51
MDR-samples): 22 (43,1%) were Fq-resistant,
25 (49%) – CAP/AM-resistant, 12 (23,5%) were
resistant to all above indicated antibiotics. Therefore
12 patients were determined as patients with XDRTB. Agreement of results between real-time analysis
and culture methods was 87% for Fq and CAP/AM
antibiotics (for Fq – 84,1%; for CAP/AM – 89,6%).
Sensitivity of real-time method as compared with
cultural method was 90%, specificity – 98% (FQ –
96%, CAP/AM – 100%).
P204
DIAGNOSIS OF PULMONARY TUBERCULOSIS
USING GENOTYPE MTBDR ASSAY IN HIVINFECTED PATIENTS, TG.-MURES, ROMANIA
Lilla Lőrinczi1, Maria Nemes2, Zaharia Kézdi
Erzsébet Iringó3, Mihaela Patraulea4, Székely
Edit5, Vas Krisztina Eszter5, Lőrinczi Zoltán6
1
Department of Microbiology, University of
Medicine and Pharmacy, Tg.-Mures, Romania;
Laboratory Tb University hospital
2
Laboratory Tb University hospital
3
Department of Infectious Diseases, University of
Medicine and pharmacy, Tg.-Mures, Romania
4
Tb University hospital, Tg.-Mures, Romania
5
Department of Microbiology, University of
Medicine and pharmacy, Tg.-Mures, Romania
6
Department of anatomy, University of Medicine
and pharmacy, Tg.-Mures, Romania
The incidence of tuberculosis (TB), especially the one
caused by multidrug resistant (MDR) Mycobacterium
tuberculosis (MTB) strains exhibits a continuous
increase in the last years, as experienced in cases
of HIv-infected patients. In Mures County, Romania
theafferentfiguresrisefromanapproximate10%in
2009 to 80% in 2012. Hence, it turned mandatory the
earlydiagnosisoftuberculosisandtheidentification
of strains causing the infection. The towering problem
was addressed using molecular biology facilities.
Material and methods: The samples were collected
between 2012 October - 2013 March, 28 sputum
samples and 10 MTB strains from 38 HIv infected
pacients, who were hospitalized at Mures Regional
93
HIv/AIDS Centre. We used GenoType MTBDR Plus
(version 2) kits for identifying the presence of MTB
DNA and the rpoB, inhA, katG genes (wild type or
mutations).
Results: 7 out of the 10 MTB strains isolated from
HIv-infected persons were MDR. From the 28
samples tested 24 were positive for MTB-DNA and
20 (83.33%) were MDR.
Conclusions: Time elapsed to diagnose the TB
infectionandidentificationofMDRstrainsusingthe
GenoTypeMTBDRpluskitissignificantlyshorterthan
using classical methods, cultivation and sensitivity
testing on Loewenstein-Jensen medium.
The very high and growing incidence on MDR MTB
strains among the HIv infected patients in Mures
County justifies the use of GenotypeMTBDR kit for
investigating the samples originating from these
patients, allowing an early diagnosis and individually
tailored medication according to the sensitivity of the
strain in cause.
correction of therapy was performed on the results of
MT. Statistical analysis was performed using EpiCalc
and chi-square criterion.
Results: patients with MDR-TB was identified as
evidenced data the MBT was isolated by the way
(MT) in 46.5% cases, (MG) method – 67.9 % (χ2
=17.47, p<0,001). 64 (50.4%) MBT cultures had
multidrug-resistant (MDR-TB) DR to R by the way
(MT) was determined in 65 (55.5%) cases, DR to H –
73 (62.4%) cases. DR to H was presented by gene
katG mutations in 61 (52.1%) cases, in gene inhA
– 17 (14.5%) and in gene ahpC – in 3 (2.6%) cases.
DR to R was determined by gene rpo B mutations
in 68(58.2%) cases. In the I group was significantly
earlier termination of bacteria within 2 months (46,2%
(I) vs. 15,7% (II), p<0,01) and closure of lung cavities
within 6 months (91,5% (I) vs. 62,8% (II), p<0,01).
Conclusion: MG allow in early assign therapy for
patients with MDR-TB in 5-7 days, which allows a
highefficiencyoftheTB-therapy.
P232
P253
THE POSSIBILITY OF EARLY CORRECTION
OF CHEMOTHERAPY FOR PULMONARY
TUBERCULOSIS ON THE BASIS OF
MOLECULAR GENETIC METHODS
Mariya Pavlova1, Viatcheslav Zhuravlev1, Ludmila
Archakova1, Nadezhda Sapozhnikova1, Natalya
Solovyeva1, Anna Starshinova1
petersburg, Russian Federation
Introduction: in department of phthisiopulmonology
in the 2010-2012 examined 187 patients (men-115,
women – 72) with newly diagnosed pulmonary TB:
infiltrative tuberculosis -74.9% (140); dissemination
lunglesiontuberculosis-18.2%(34);fibro-cavernous
TB - 6.9% (13).
Objectives: ‘determine the effectiveness of therapy in
rapiddiagnosticsdrugresistanceinthefirstidentified
patients with pulmonary tuberculosis.
Methods: sputum from 187 TB patients was
simultaneously analyzed using microbiological
tests (MT) and molecular-genetic methods (MG).
Microbiological tests included culturing on solid
medium (Lowenstein-Jensen), liquid medium
Middlebrook 7H9 utilizing BACTEC MGIT 960 with
determination of drug resistant (DR) of mycobacterium
tuberculosis (MBT) using absolute concentration
method. MG was used: real time polymerase chain
reaction(RTPCR)–forMTBcomplexidentification
in clinical specimens and DNA microarray analysis –
TB biochip (TBCh) – for drug resistance (DR) testing
directly in the clinical specimens. TBCh identifies
mutations in four MBT genes associated with DR
to rifampin (R) in gene rpoB and to isoniazid (H) katG, inhA, ahpC genes. The patients divided into
two groups: I group (n=117) - correction of therapy
was performed on the results of MG; II group (n=70) -
94
THE CORRELATION BETWEEN PHENOTYPIC
AND GENETIC RESISTANCE TO ETHAMBUTOL
IN PATIENTS FROM MOSCOW REGION
Ruslan Ludannyy1, Maria Alvarez Figueroa2,
Anastasiya Prokopenko1
1
Federal Budget Institute of Science “Central
Research Institute for Epidemiology” of Federal
Service on Consumers’ Rights protection and
human Well-Being Surveillance, Moscow, Russia
2
Moscow Scientific and Clinical Antituberculosis
Centre, Moscow government health Department,
Russia
Objectives: Ethambutol (E) is one of the first-line
drugs and has an impact on cell’s pathway. Basically
E inhibits mycobacterial arabinosyl transferases
encoded by the embCaB operon which contains three
genes (embC, emba and embB). Nowadays SNPs in
306 codon are the most frequently used markers for
E resistance detection.
Aim: The aim of our investigation is to determine
the spectrum of mutations of embB gene which are
associated with E resistance and to find correlation
between phenotypic and genetic MTC resistance
among patients from Moscow region.
Methods: A total of 118 clinical MTC isolates were
collected in 2008 – 2009 from patients of Clinical
TB Hospital #7 (Moscow, Russia). The LöwensteinJensen absolute concentrates culturing method was
applied for the detection of phenotypic resistance to
E (2 mg/ml). The method of Sanger sequencing was
used for the detection of genetic resistance in EmbB
gene.
Results: Thepolymorphicsiteswereidentifiedin98
isolates (69 sensitive and 29 resistant) in 936 b.p. part
of EmbB gene where 97% of previously described
mutations were concentrated. Consequently, 51
isolates had only one non-synonymic mutation; 5
isolates had more than one change and 3 isolates
revealed combination of mutant and wild types. 39
isolates had no alterations; however, two of them
had phenotypical resistance. Overall, 19 types of
mutations were observed during research. Three of
nineteen mutations were synonymic and have not
been described previously: p. Ser261Ser, p. Gly294Gly
and p. Ala453Ala. The most widely represented SNPs
were: p. Met306val – 31.6%, p. Met306Ile – 24.5%,
p. Gly406Ser – 8.2% and p. Asp354Ala – 7.1%. The
correlation between phenotypic and genetic sensitivity
was 43% for phenotypic resistant isolates and 53% for
sensitive one’s. Two phenotypical resistance isolates
were studied more thoroughly because genetic
mutations had not been observed. For these samples
we had analyzed the total EmbB gene sequence,
however, no mutations were found.
Conclusion: In our opinion the role of emb306
SNP in clinical diagnostics of resistance detection
is overestimated because we have found it only in
53.1% of all cases. The discordant results that have
been obtained in the course of our study demonstrate
that E resistance of M. tuberculosis complex is related
to mutations located outside of EmbB gene and that
novel mutations contributing to E resistance are likely
to be discovered.
P256
MDR TB WITH MIxED STRAINS: A CHALLENGE
IN DIAGNOSTIC AND TREATMENT OF PATIENTS
Valeriu Crudu1, Elena Romancenco2, Ecaterina
Noroc3, Nadejda Turcan3, Liliana Domente3, Sofia
Alexandru3, Dumitru Chesov3, Stefan Niemann4,
Matthias Merker4, Sabine Rüsch-Gerdes4,
Antonino Catanzaro5
1
Center for health policies and Studies,
Chisinau, Moldova
2
State Medical and pharmaceutical University,
Chisinau, Moldova
3
phthisiopneumology Institute, Chisinau,
Moldova
4
national Reference Center for Mycobacteria,
Forschungszentrum Borstel, Borstel, germany
5
Department of Medicine, University of
California, San Diego, USa
Rationale: Although progress has been made
to reduce global incidence of tuberculosis, the
emergence of multidrug-resistant (MDR) tuberculosis
during the past decade threatens to undermine these
advances. The occurrence of mixed infections of M.
tuberculosis is no longer disputed. However, their
frequency, and the impact they may have on our
understanding of TB pathogenesis and epidemiology,
remains undetermined.
Re- infections with MDRTB strains may occur in
settings where the infection pressure is high, long
period of treatment and low infection control in hospital.
The magnitude and reasons of TB nosocomial
transmission are only insufficiently investigated,
especially in developing countries with high rates of
MDRTB.
Objectives: This study aimed to describe the level
of mixed infection among MDRTB patients and the
possibilities of MTBDRPlus test to evaluate the
reinfection and mixed strains.
Methods: The MTBDR Plus test results from TB
patients examined before and during follow-up of
treatment were evaluated. In total 271 results from
193 New TB cases and 78 patients with re-treatment
cases were assess. The RIF Sensitive results was
determine in 38.7%, the RIF resistance – in 38.0%,
in 14.0% of cases the results was indeterminate and
9.2% negative results. The INH Sensitive results was
determined in 33.6%, the INH resistance – in 39.1%,
in 17.3% of cases the results was indeterminate and
10.0% negative results. The results of selected 64
patients, who were before treatment sensitive and
became MDRTB during follow-up treatment, were
compared. Among patients with follow-up treatment
the level of indeterminate results of MTBDRPLus
was very high – 48.4% for RIF and 57.8% for INH.
The indeterminate results can be caused by mixed
infection, occurred during in-patient treatment. The
high level of nosocomial re-infection in these setting
was proven in studies performing before (v. Crudu,
2011). Genotypic analysis was done (IS6110 DNA
fingerprinting,MIRU)forconfirmedthemixedinfection
from patients with indeterminate results of LPA. The
mixed infection was proven in 67.0% for RIF and
69.0% for INH.
Conclusions: We have shown that, among patients
with MDR TB, mixed infection may be caused by
re-transmission between patients during in-patients
treatment. The “indeterminate results” from MDRTB
patients, obtained by MTBDRPlus test, can indicate
the possible re-infection and mixed strains.
Acknowledgement: This study was funded by NIAID
(u01AI082229, PI: A.Catanzaro) through the Global
Consortium for Drug-resistant TB Diagnostics.
P259
ExTERNAL qUALITY CONTROL FOR DRUG
SUSCEPTIBILITY TESTING IN ROMANIAN
TUBERCULOSIS LABORATORY NETWORK
Daniela Homorodean, Andreea Melinda Jodal
national Reference Laboratory, Cluj napoca,
Romania
The quality of drug susceptibility tests (DST) results
are crucial for the programmatic management of drug
resistant tuberculosis (TB) cases. In Romania there
are 40 county laboratories (labs) and two National
95
Reference Laboratories (NRL) performing annually
about12,500DSTforfirstlinedrugsIsoniazid(INH)
and Rifampicin (RMP).
Aim: Assessment of the DST external quality control
(EqC) results for INH and RMP in the 42 laboratories
of the network performing this test in two successive
rounds.
Material and methods: For 2009 and 2012 EqC
rounds we used the international M. tuberculosis
panel of 20 strains received from the Supranational
Reference Laboratory Stockholm. Strains were
sub-cultured, renumbered and coded and were
transported to the labs by an authorized company.
Participants were informed about risk infection and
the necessity to handle specimens in bio-safety
conditions. Absolute concentration method was used
for DST. Results were reported in forms which were
sentwiththestrains.Sensibility,specificity,accuracy
(concordant results) and predictive values of the
results were calculated.
Results: In 2009 round all labs obtained 100%
concordant results for RMP and 95.2% (40/42) for
INH. One false susceptible (FS) INH result was
reported in one lab, and another lab reported one
FS INH and one false resistant (FR) INH result. In
2012 round, for RMP, 100% concordant results were
obtained in 47.6% (20/42) of labs, 95% concordant
results in 45.2% (19/42) of labs, 90% concordant
results in one lab and 85% concordant results in 2
labs. Errors consisted in FR RMP results. The errors
were not repeated in the same labs in the two rounds.
For the labs which obtained unsatisfactory results we
recommended the training of the staff in one NRL.
Conclusions: The results obtained in the two EqC
rounds are good and reliable. In order to maintain
and document the high quality of DST results it is
necessary to yearly repeat DST EqC.
P262
LATE-PCR DETECTION OF M(x)DR-TB USING
FLUORESCENT SIGNATURES
L. Rice,1 J.Rice1, L. Wangh1; N. Kurpina2, B.
Kreiswirth2, N. Casali3, F. Drobniewski3; M. De
Vos4, R. Warren4
1
Dept. of Biology Brandeis Univ., Waltham, Ma,
USa
2
phRI, newark, new Jersey, USa
3
Blizard Institute, Queen Mary College, University
of London, London, UK
4
DST/nRF Centre of Excellence for Biomedical
Tuberculosis Research, Stellenbosch Univ.
Tygerberg, South africa
Background: We have constructed and are
evaluating a highly informative, rapid, single-tube
assay for M(X)DR tuberculosis using technologies
invented at Brandeis university.
Methods: AmplificationoccursbyLATE-PCR,which
96
generates single-stranded amplicons. Detection
occurs by Lights-On/Lights-Off probes, which makes
it possible to scan long DNA targets for mutations. All
amplicons are analyzed simultaneously at end-point
at temperatures below the annealing temperature.
quasar: Rifampin resistance (rpoB gene); Cal Red:
Isoniazid, Ethambutol, Fluoroquinolone resistance
(inhA promotor, embB gene, gyrB gene); Cal Orange:
Isoniazid and Fluoroquinolone resistance (katG gene,
gyrA gene); Fam: Aminoglycoside and Streptomycin
resistance (rrs1401, rrs904-908, rrs504-516). If a 5th
color channel is available it can be used to detect
additional targets of interest. An internal control for
quantitative analysis, temperature mark, and primer
specificityisalsopresent,labeledinCalOrange
Current Results: Unique “fluorescent signatures”
were obtained for all different rifampicin-resistant
strains of M. tuberculosis tested, plus several drugsensitive strains containing different neutral of
mutations. Each of the predominant mutations for the
other drug resistant targets has its own fluorescent
signature, color and temperature space. This assay
is also specific for MTBCs and does not give false
positives with any NTMs. This assay is robust and
detects as little as a single bacterial genome in a
background of 10,000 human genomes. No false
positives or negatives have been observed thus far.
Conclusions: This single-tube M(X)DR-TB assay
is just one of many possible assay designs that can
be constructed using the suite of Brandeis university
technologies. Supported by the TSB (uK), Smiths
Detection Diagnostics, Inc., Brandeis university, and
NIH. This and several related assays are now under
development for Hain Lifescience.
P161
EFFICIENCY OF CHEMOTHERAPY OF xDR TB
PATIENTS WITH LINEZOLIDE
Anastasia Samoilova, Irina Vasilyeva, Tatev
Bagdasarian, Marina Burakova, Svetlana
Moiseyeva
Central TB Research Institute, Russian academy
of Medical Sciences, Moscow, Russia
Objective: ToassessefficiencyoftreatmentofXDR
TB patients with linezolide.
Methods: Treatment of 112 XDR TB patients, enrolled
in this study, consisted besides chemotherapy
of collapse therapy (artificial pneumotorax and/
or pneumoperitonium), immunotherapy, additional
albuminous nutrition if necessary, supporting therapy
aimed at prevention of possible side effects. All XDRTBpatientsweretreatedbycapreomycin,moxifloxacin,
pyrazinamide, cycloserine, PAS, protionamide and/
or ethambutol and amoxicillin+clavulanic acid or
clarithromycin. Patients were divided into three
groups: the 1st group included 44 patients who
received complex treatment and linezolide in the
dose of 600 mg per day during 4 months and the 2nd
group - 34 patients who received complex treatment
and linezolide in the dose of 600 mg per day during
12 months and the 3rd group consisted of 34 patients
receiving the same complex treatment without
linezolide. XDR of M. tuberculosis was diagnosed by
culture method on liquid media with BACTEC MGIT
960. Therapy results were assessed in 12 months by
frequency of negativation of M. tuberculosis with a
culture method and by clinical and X-ray dynamics of
a treatment process.
Results: Culturemethodconfirmedthat34(77,3%)
patients became cultures negative in 12 months in the
1st group, 33 (97,1 %) - in the 2nd group and only 12
(35,3%) patients in the 3rdgroup(р<0,05).In1st group
in 12 months 18 (40,9%) patients showed healing of
cavities, in the 2nd group – 19 (55,9%) and in the 3rd
group - 8 (23,5%) patients, p1,3<0,05, p2,3<0,05.
Conclusion: Long-term administration of linezolide
in the regimen of chemotherapy for XDR TB patients
significantlyincreasesefficiencyoftheirtreatment.
97
TB BIOMARKERS AND NOVEL VACCINE STRATEGIES
P21
DRUG TOLERANCE TO ETHAMBUTOL OF
MyCOBaCTERIUM TUBERCULOSIS CELL WALL
DEFICIENT L-FORMS
Georgi Slavchev, Nadya Markova
Institute of Microbiology, Bulgarian academy of
Sciences, Sofia, Bulgaria
The loss of cell wall in mycobacteria results in
appearance of highly pleomorphic forms with new
biologic characteristics, known as L-forms. It is known
that L-forms occur along with resistance to factors
that trigger their appearance and survive unfavorable
conditions much longer than the classic bacteria. In
this respect, it was of interest to study the relationship
between L-form formation in M. tuberculosis and
exhibition of drug tolerance to Ehambutol (EMB) which
acts through the inhibition of cell wall synthesis.
Model using in vitro cryogenic stress treatment and
subsequent cultivation in two variants of Middlebrook
7H9 semisolid medium (EMB containing medium
in concentration of 2mg/l and control medium) was
used for induction and selective isolation of L-forms
both from EMB sensitive (No18 ) and resistant (No
43) strains of M. tuberculosis. The obtained L-form
variants of tested strains gave rise to colonies with
typical “fried eggs” shape. Electron microscopy
showedspecificmorphologyandultrastructureofcell
wall deficient bacterial population. EMB resistance/
susceptibility was evaluated phenotypically and by
PCR assay targeting embB 306 mutations. It was
found that L-forms originating from a sensitive No18
strain and obtained after cultivation in both variants
of semisolid medium (with and without EMB) showed
phenotypically tolerance to high concentration of EMB
(16mg/l). It is interesting to note that amplification
of embB 306 failed in all L-form variants of both
M. tuberculosis strains, although all L-forms were
confirmedbyIS6110RT-PCR.
We suggest that loss of specific targets for EMB in
L-forms of M. tuberculosis may underlie the phenotypic
drug tolerance to this drug.
Acknowledgements This work was supported by
grantIDNo.02/27oftheNationalScientificFundin
Bulgaria
P86
MALARIA, TUBERCULOSIS, HIV and HBV
COINFECTIONS IN A RURAL POPULATION IN
THE SOUTH OF GHANA
98
Natalia Julca1, Alejandra Martinez-Serna2, Maria
Carmen Menendez1, Carolina Garrido4, Patricia
Marin-Garcia3, Antonio Puyet2, Jose Manuel
Bautista2, Carmen De Mendoza3, Maria Jesus
Garcia5, Amalia Diez2
1
Universidad autonoma de Madrid, Madrid, Spain
2
Universidad Complutense de Madrid, Madrid,
Spain
3
Universidad Complutense de Madrid, Madrid,
Spain; Universidad Rey Juan Carlos, Alcorcón,
Madrid, Spain
4
hospital Carlos III, Madrid, Spain
5
Introduction: The clinical course of patients infected
by Plasmodium falciparum in a region highly endemic
for malaria could be influenced by the co-infection
with other main pathogens, such as M. tuberculosis,
HIv and HBv. In this scenario, the internal competition
among pathogens not only to each other but also with
the immune system could determine the prognosis
and outcome of the diseases.
Objective: To study the detection of single and
multiple infections in a high prevalent region of
the four main pathogens: Plasmodium falciparum,
HumanImmunodeficiencyVirusandM. tuberculosis
and Hepatitis B, aiming to determine the level of coinfection in a rural population in the South of Ghana.
Methods: Dried Blood Spots, as well as sera were
collected from patients attending a Hospital of Asikuma
(Ghana). Detection of Plasmodium was performed by
PCR-quantificationofparasiteDNAfromthespots.
Total nucleic acids of mycobacteria were isolated
from the spots. Mycobacterial DNA and cDNA were
detected following published procedure (Cubero et al,
2012).
HIv and HBv chronic infections were analysed by
thedetectionofspecificantibodiesusinganELISA.
In order to determine HBv chronic infection the
presence of positive HbsAg was required.
Results: A total of 58 patients were analysed (15
male and 43 female, 22 known to be pregnant). The
mean age was 23 y.o., only three patients were over
37 y.o.
Overall, at least one of the pathogens was detected
in 79.3% of patients. DNA from P. falciparum was
the commonest detected as single pathogen (22%)
followed by detection of mycobacterial RNA (19%)
corresponding to M. tuberculosis in 2/3 of the cases.
Co-infection of HBv or P. falciparum with mycobacteria
was also frequent (8.5% and 17% respectively). Non
tuberculous mycobacteria were more frequently
detected than M. tuberculosis in those co-infected
samples.
up to three different pathogens were detected in four
patients. Remarkably, the two cases with positive
detection of HIv belong to this group.
Conclusions: Multiple infections was seen, being
co-infection of P. falciparum and M. tuberculosis the
most common. The knowledge of the presence of
multiple infections could be important to determine
actions to be made for the management of patients
with Malaria.
Funding resources /Acknowledgements
Supported by Spanish Ministry of Innovation and
Science (BIO 2010-17039)
P205
CIRCULATING MIRNA SIGNATURES IN
TUBERCULOSIS
Paolo Miotto1, Grace Mwangoka2, Ilaria C.
Valente1, Luca Norbis1, Giovanni Sotgiu3,
Alessandro Ambrosi4, Luigi Codecasa5, Delia
Goletti6, Alberto Matteelli7, Klaus Reither8,
Daniela M. Cirillo1
1
Italy
2
Bagamoyo Research and Training Centre /
Ifakara health Institute, Bagamoyo, United
Republic of Tanzania
3
Epidemiology and Medical Statistics Unit,
Department of Biomedical Sciences, University
of Sassari, Sassari, Italy
4
San Raffaele University, Milan, Italy
5
Regional Reference Center for Tb “villa Marelli”,
niguarda Ca’ granda hospital, Milan, Italy
6
National Institute of Infectious Diseases;
Department of Epidemiology and preclinical
Research; National institute for Infectious
Diseases “L. Spallanzani”, Rome, Italy
7
Institute of Infectious and Tropical Diseases,
University of Brescia, Brescia, Italy
8
Swiss Tropical and public health Institute
(Swiss Tph), Basel, Switzerland
Early diagnosis and treatment are key elements to
achieve tuberculosis (TB) control. However, current
diagnostic tools suffer of several limitations, especially
for those at higher risk of TB disease and death, such
aschildrenandimmunodeficientindividuals.Recent
studies showed that circulating microRNA (miRNA)
can be successfully used as biomarkers for several
physiological and pathological statuses, including
TB.
This study aims to identify the different serum miRNA
profilesassociatedwithM.tuberculosisinfectionand
disease.
For the study we enrolled healthy controls (H),
subjects with active pulmonary TB (PTB), latent TB
(LTBI), extra-pulmonary active TB (EPTB), patient
with pulmonary infections other than TB (OPI). The
subjects included in the study were enrolled in 2 sites
in Italy and 2 sites in Africa (uganda and Tanzania).
To minimize individual variation, sera from 10 subjects
within the same category were pooled. The miRNA
profile in total RNA was analyzed by TaqMan LowDensity Array (TLDA) (Applied Biosysistem). We
also evaluated single-subject miRNA profiles in 18
PTB and 18 H sera from the European Region and
10 PTB and 10 H sera from the African population
by TLDA. quantile normalization was performed for
each dataset (pools/individuals), and Student’s t-test
was used to compare the levels of circulating miRNAs
between categories.
Out of the 672 miRNAs analysed, 114 showed to
be differentially up- or down-regulated within the
categories analyzed (P<0.05). In particular, 38
miRNAs were differentially expressed between PTB
and H; additional 27 miRNAs were discriminatory
between PTB and OPI categories. Analysis performed
on single patients confirmed 3 miRNAs for both the
European and the African cohorts. Additionally, 4
miRNAspreviouslyunidentifiedbythepoolsanalysis
werefoundtobesignificantlydiscriminatorybetween
PTB and H subjects in both the European and African
individuals. Concluding, a signature of 7 miRNAs (3
European-specific,3African-specificand4common)
wasidentifiedasdiscriminatory.
Our study suggests that altered levels of serum
miRNAs have great potential to serve as noninvasive biomarkers for early detection of pulmonary
TB disease.
P207
IDENTIFICATION OF ROUTINE CLINICAL
MYCOBACTERIA ISOLATES WITH MALDI-TOF
MS USING THE NEW BRUKER MYCOBACTERIA
LIBRARY
Gorkem Yaman, Cigdem Celikkan, Derya Turk
Duzen Laboratories group, Istanbul, Turkey
Objective: Mycobacteria identification of routine
clinical isolates is mainly based on molecular
methods which are highly technical, time consuming
and expensive. The use of MALDI-TOF MS for
mycobacteria identification is promising; however,
despite there are mycobacterial spectra present in
the IvD databases, the results obtained with standard
preparation techniques and routine database were not
satisfactory.In this study the performance of MALDITOF MS for identification of clinical mycobacteria
strains with the new proein extraction method and the
Bruker mycobacteria library was evaluated.
Methods: A total of 145 mycobacteria isolates from
various clinical materials requested for TB culture were
included in the study. The specimens were cultured in
both BD MGIT bottles and LJ medium. Growths on
eithermediumwereEZNstainedandidentifiedifacid
fast bacilli are present. All of the isolates were initially
99
identified with PCR-RFLP analysis (PRA) using
hsp65 TB11 and TB12 primers. For MALDI-TOF MS
analysis, the mycobacterial colonies were inactivated
with ethanol, heated up to 100oC and zirconia/silica
beads were used for mechanical disruption and
finallyproteinextractionwaselicitedwithacetonitrile
and formic acid. The samples were pipetted onto
steeltargetplateandidentifiedwithBrukerMicroflex
LT system using the new Bruker mycobacteria library.
Results: The
mycobacterial
isolates
were
distributed as 75 M. tuberculosis complex and 70
non-tuberculosis mycobacteria (NTM) of which 11
M.abcessus, 8 M.fortuitum, 8 M.avium, 6 M.simiae, 5
M.intracellulare, 6 M.gordonae, 2 M.avium complex,
1 M.kansasii, 1 M.peregrinum, 1 M.porcinum/M.
septicum, 1 M.smegmatis with PRA. The remaining
20 isolates were not accurately identified and
accepted as unidentified NTM species. From the
125 accurately identified isolates, the mycobacteria
library correctly identified 107 (85,6%) to species
level. Since the mycobacteria library contains only
mycobacterial strains, the remaining 18 results were
considered as discrepant which will be subjected to
16S rRNA sequencing. The Bruker mycobacteria
library gave a spectral score higher than 1.7 for 84
(67,2%) isolates and 81 (96,4%) of these isolates
werecorrectlyidentifiedtothespecieslevel.
Conclusion: The new preparation method and the
mycobacteria library significantly increased the
number of correctly identified clinical mycobacteria
isolates compared to the IvD database. MALDI TOFMS provides fast and accurate results especially
with spectral scores higher than 1.7 using the new
mycobacteria library and it could be implemented for
routineidentificationofmycobacteriaintotheworkflow
of clinical microbiology laboratories.
P222
EVALUATION OF FLUOROTYPE MTB FOR
DIRECT DETECTION OF MyCOBaCTERIUM
TUBERCULOSIS COMPLEx AND GENOTYPE
MTBDRPLUS FOR DETERMINING OF
RIFAMPICIN AND ISONIAZID RESISTANCE
Gonca Erkose Genc, Dilek Satana, Esra Yildrim,
Zayre Erturan, Yildiz Yegenoglu, Meltem Uzun
Istanbul University, Istanbul Faculty of Medicine,
Department of Medical Microbiology, Istanbul,
Turkey
Background: The commercially available assay
FluoroType MTB (Hain Lifescience, Nehren,
Germany) is a PCR based assay for direct detection
of Mycobacterium tuberculosis complex (MTBC) DNA.
The assay principally based on targeting the IS6110
element. Amplification and detection takes place
in a FluoroCycler (Hain Lifescience) using melting
curve fluorescence Hybeacon-probe technology
in about three hours. GenoType MTBDRplus (Hain
100
Lifescience) test is based on DNA strip technology
for the identification of MTBC and its resistance to
rifampicin (RMP) and isoniazid (INH). The aim of this
study was to evaluate the performance of FluoroType
MTB for rapid determination of MTBC and the
performance of GenoType MTBDRplus for detection
of RMP and INH resistance in clinical samples.
Methods: Totally 247 (124 respiratory, 123
nonrespiratory) samples were decontaminated,
neutralized and concentrated with conventional
methods. Processed samples were separated into
twovolumes.Thefirstvolumewasusedforacid-fast
bacillus (AFB) microscopy, culture in Bactec MGIT 960
liquid medium and Löwenstein-Jensen solid medium.
Second volume was used for analysis by FluoroType
MTB. GenoType MTBDRplus was performed for the
samples which were found to be positive for MTBC
detection.
Results: Twenty three of 247 samples (9.3%) were
positive both by culture and FluoroType MTB, while
12 (4.8%) of these samples were AFB negative.
FluoroType MTB failed to detect MTBC in a gastric
lavage which were culture positive. For this assay
overall sensitivity, specifity, positive and negative
predictive value were determined as 95.8%,
100%, 100% and 99.5% respectively. GenoType
MTBDRplus was performed on 23 clinical specimens
which were positive by FluoroType MTB. A valid
result could not have obtained for three specimens
(a biopsy, a urine and a gastric lavage), while 20 of
them were found susceptible to RMP and INH. For
three specimens, result was only available when the
test was performed with cultivated samples. It could
may be due to the clinical specimens which contained
number of mycobacteria under the detection limit of
the test. Antituberculous drug susceptibility test was
performed for 23 strains by Bactec MGIT 960 method
and all strains were found susceptible to RMP and
INH.
Conclusion: FluoroType MTB assay provided quick
and reliable direct detection of MTBC from clinical
specimenswithhighsensitivity(95.8%)andspecifity
(98.7%). The performance of GenoType MTBDRplus
was determined to be problematic in some occasions
regarding to the count of mycobacteria in clinical
samples, but it is found to be a reliable test for
cultivated samples.
NONTUBERCULOUS MYCOBACTERIAS
P18
IN MExICAN MESTIZOS THE HLA-DRB1*01
ALLELE CONFERS GENETIC SUSCEPTIBILITY
TO LEPROMATOUS LEPROSY (LL)
Monica Escamilla-Tilch2, Iris Estrada-García1,
Nora M Torres-Carrillo3, Rosalío Ramos-Payán4,
M. Isabel Salazar1, Mary Fafutis5, Roberto
Arenas-Guzmán6, Sergio Estrada Parra6, Julio
Granados7
1
Dept. de Inmunol., Esc. nal. de Ciencias
Biológicas, Ipn., México City, México
2
Dept. de Inmunol., Esc. nal. de Ciencias
Biológicas, IPN, México City, México; Dept. de
Transplantes, Inst. nal. de Ciencias Médicas y
nutrición Salvador zubirán, México City, México
3
Departamento de Investigación Básica, Instituto
nacional de geriatría, Secretaría de Salud,
México City, México
4
Facultad de Ciencias Químico-Biológicas, UaS,
Culiacán, Sinaloa, México,
5
Lab. de Inmunol., Inst. Dermatol. de Jalisco
José Barba Rubio, guadalajara, Jalisco, México,
6
Sec. de Micol., hosp. gral. Dr. M. gea gonzález,
México, D.F.
7
Dept. de Transplantes, Inst. nal. de Ciencias
Médicas y nutrición Salvador zubirán, México
City, México
Despite great efforts from the National Leprosy Control
Programme, leprosy remains endemic in some
regions of México. There is a great heterogeneity
both in geographical distribution of cases, and in
Mycobacterium leprae genotypes. Reasons for these
differences are unclear, but may reflect the genetic
heterogeneity of the Mexican Mestizo population and
the introduction of the infectious agent several times
from different geographical areas of the world. Recent
studies indicate that pathogenesis may be a two-step
process in which a group of genes controls susceptibility
to leprosy per se, while a different set of genes controls
the clinical manifestations of the disease. Regarding
genes in chromosome region 6p21, studies have
shown association between leprosy susceptibility
and both Major Histocompatibility Complexes (MHC),
class I and class II; in particular for locus DRB1 of the
MHC class II that participates on antigen processing
and presentation to T lymphocytes, being crucial for
T cell-mediated immunity. Considering HLA genes
influencesusceptibilitytowardsleprosy,aswellasthe
clinical form LL or tuberculoid (TT), we investigated
the association of HLA-DRB1 alleles in 52 Mexican
patients (39% female and 61% male, mean age 65±14
years, range 18-86 years) from eight states, 41 with
LL. 99 healthy individuals (50% female and 50% male,
mean age 40±10 years, range 28-52 years) were
included as controls. We found that HLA-DRB1*01
allele in a Mexican mestizo population with leprosy
(n=52)issignificantlymorefrequent(23.00%,pc<
0.001, OR = 5.60) than in healthy individuals (n = 99,
5.00%).ThisisthefirsttimetheassociationofHLADRB1*01 with immune susceptibly to lepromatous
leprosy is described in this population.
P41
MYCOBACTERIA IDENTIFICATION FROM
PATIENTS WITH RESPIRATORY SYMPTOMS
ADMITTED IN PEDRO KOURÍ INSTITUTE,
HAVANA, CUBA
María Rosarys Martínez Romero1, Misleidis
Sardiña2, Grechen García2, Lilian María Mederos2,
Yoandra Abad3
1
pedro Kourí Institute, havana, Cuba
2
national Reference Laboratory in Tuberculosis
and Mycobacteria, pedro Kourí Institute, havana,
Cuba
3
Mathematical Modeling. Epidemiology
Departmen. pedro Kourí Institute, havana, Cuba
Background:
Mycobacteria
are
responsible
for high morbidity and the rapid diagnosis is
essential to implement an effective control.
Aim: To perform the isolation and identification of
mycobacteria in patients with respiratory symptoms.
Methods: We studied 942 patients with respiratory
symptoms. Samples were processed at the National
Reference Laboratory of tuberculosis (IPK), from
January to December 2012. The samples processing
and culture, was done by manual laboratory
procedures. The positive culture for acid-fast
bacilli, were performed a conventional biochemical
identificationtestsandrapidtest(SDtestBIOLINE)
to differentiate the complex M. tuberculosis and
nontuberculous mycobacteria (NTM), simultaneously.
TheclassificationofNTMwasdoneaccordingtothe
established procedures.
Results: The rapid test to differentiate M. tuberculosis
andNTM,showed100%ofsensitivityandspecificity.A
55.7% of the isolates corresponded to M. tuberculosis
and 44.3% were NTM; within them M. aviumintracellulare (MaI) and M. malmoense were the most
frequent (21.4% and 11.4%, respectively). Analysis by
type of sample (pulmonary and extrapulmonary) and
isolated species showed to MaI and M. malmoense
an OR of 8.19 and 5.22 and p values 0.05. When
analyzing the results by type of mycobacteria isolated
101
and suffer or not HIv, we obtained an OR of 7.61 (p
= 0.0002).
Conclusions: SD Bioline test was adequate
method to differentiate M. tuberculosis and NTM.
TheMNTareresponsibleforasignificantnumberof
infections by mycobacteria and MaI being the most
common isolation. MaI and M. malmoense isolates
were found more frequently in extrapulmonary
samples. Patients with HIv were risk 7 times
greater than non-HIv to suffering tuberculosis.
P51
A CASE OF MyCOBaCTERIUM TERRaE
INFECTION IN CATTLE IN BOSNIA AND
HERZEGOVINA
Hajrudin Besirovic1, Amer Alic1, Silvio Spicic2,
Zeljko Cvetnic2, Senad Prasovic1
1
University of Sarajevo, veterinary Faculty,
Department of pathology, Sarajevo, Bosnia and
herzegovina
2
Croatian veterinary Institute, zagreb,
Department of Bacteriology and
parasitology,Laboratory for Bacterial zoonosis
and Molecular Diagnosis of Bacterial Diseases,
zagreb, Republic of Croatia
Non-tuberculous Mycobacteria (NTM) are worldwide
opportunist pathogens which can infect both humans
and animals. Infections with these mycobacteria can
have various clinical manifestations and may cause
allergic sensitisation of the infected animals. Besides
the potential zoonotic risk these infections in cattle can
interfere with intradermal tuberculin test and hamper
the in vivo diagnosis of bovine tuberculosis resulting
in significant economic losses due to unnecessary
restrictions and culling of reactor animals. Here we
describe a case of Mycobacterium terrae infection in
a cow on a small dairy household in Central Bosnia
and Herzegovina.
For the identification of the disease and causative
agent comparative tuberculin skin test (TST),
pathomorphology, microbiology and molecular
methods were applied.
At necropsy of a cow that was positive on annual
routine tuberculin skin testing, severe multifocal
granulomatous dermatitis and paniculitis were
observed. Furthermore, moderate granulomatous
(parasitic) colitis was also noted. Histopathology
revealed granulomatous dermatitis. Ziehl-Neelsen
staining was negative for acid fast bacteria. Regional
and thoracic lymph nodes, and skin lesions were
submitted for microbiology examinations. Isolated
bacteria were identified by molecular methods as
Mycobacterium terrae.
Our findings confirms the difficulties that nontuberculous mycobacteria can cause in the in vivo
diagnosis of mycobacterial infections, especially in
conjunction with parasitic infestations.
102
Keywords: Cattle, Non-tuberculous Mycobacteria,
Mycobacterium terrae, Bosnia and Herzegovina
P58
COMPARATIVE In vITRO ACTIVITIES OF
RIFAMPIN, RIFAPENTINE AND RIFABUTIN
AGAINST MyCOBaCTERIUM KanSaSII.
Michael Cynamon, Mary Sklaney
vamc, SUny Upstate Medical University,
Syracuse, new york, United States
The currently recommended regimen for M. kansasii
infection is isoniazid (INH), rifampin (RIF), and
ethambutol (EMB) for 12 – 18 months. RIF is the
key agent in this regimen. EMB likely decreases
the development of resistance to RIF. It is unclear if
INH plays a significant role since its in vitro activity
against M. kansasii is quite poor. The purpose of the
present study was to evaluate the comparative in
vitro activities of RIF, rifapentine (RPT), and rifabutin
(RBT) against clinical isolates of M. kansasii.
RIF, RPT, and RBT were obtained from Sigma
Aldrich Chemicals (St. Louis, MO), Merrell Dow
Pharmaceuticals (Cincinnati, OH), and Adria
Laboratories (Dublin, OH) respectively. These
compounds were dissolved in DMSO to 1mg/ml prior
to freezing at -200C. They were tested in Middlebrook
7H10 broth, pH6.6 (7H10 agar formulation with
agar and malachite green omitted) supplemented
with 10% Middlebrook oleic acid-albumin-dextrosecatalase enrichment and 0.05% Tween 80 at 1µg/
ml – 0.001µg/ml in polystyrene 96-well round-bottom
microtiter plates. To each well, 50µl of the appropriate
mycobacterial cell suspension was added to yield a
finalconcentrationofabout1x105CFu/ml (range for
various isolates tested was 1.6 x 104 – 3.0 x 106 CFu/
ml). Plates were incubated at 370C in ambient air for
7 - 10 days prior to reading.
The MIC50/MIC90 (µg/ml) of RIF, RPT, and RBT were
0.06/0.125, 0.015/0.125, and 0.004/0.015 respectively.
RBTwassignificantlymoreactivein vitro than either
RIF or RPT.
It would be interesting to evaluate the comparative
activities of these compounds in a murine model of M.
kansasii infection to determine whether a rifamycin
otherthanRIFwouldbepotentiallymoreefficacious
for treatment of M. kansasii infections in humans.
P82
MyCOBaCTERIUM BRUMaE TRIGGERS CELLCYCLE ARREST IN BLADDER CANCER CELLS
Silvia Secanella-Fandos1, Gemma OrpellaAceret1, Eduard Torrents2, Alejandro SánchezChardi3, Julia Lorenzo4, Manuela Costa5, Marina
Luquin1, Esther Julián1
Dept. genètica I Microbiologia. Universitat
autònoma de Barcelona, Bellaterra, Spain
2
Dept. Cellular Biotechnology. Institute for
Bioengineering of Catalonia – Ibec, Barcelona,
Spain
3
Servei de Microscopia. Universitat autònoma de
Barcelona, Bellaterra, Spain
4
Institut de Biotecnologia I Biomedicina.
Universitat autònoma de Barcelona, Bellaterra,
Spain
5
Unitat de Citometria. Universitat autònoma de
1
Mycobacterium bovisBCGisthefirstoptiontreatment
for high-risk non-invasive bladder cancer patients.
Although the exact mechanism by which BCG acts is
not completely known, it has been demonstrated that
BCG exerts both a direct antitumor effect by inhibiting
tumor proliferation, and an indirect antitumor effect by
stimulating patient immune cells, which in turns kill
tumor cells. Previous studies have shown that BCG
inhibits tumor cell proliferation by inducing apoptosis
and cell cycle arrest. We have demonstrated that the
non pathogenic Mycobacterium brumae also inhibits
tumor bladder cell proliferation. In the present work
we aimed to elucidate how M. brumae is able to inhibit
tumor growth.
Bladder cancer cells (T24 cells) were infected with
BCG or M. brumae (MOI 10:1). Forty-eight hours after
infection, apoptosis was measured in cell cultures by
usingdifferenttechniques:1)flowcytometrytoquantify
early apoptosis after labeling the cells with annexin v/
propidium iodide; 2) agarose gel electrophoresis to
analyse apoptotic DNA fragmentation from infected
cells: 3) ELISA to detect activation of caspase 3;
andfinally,4)bothtransmission(TEM)andscanning
electron microscopy (SEM) to observe ultrastructural
changes in morphology (membrane blebbing,
fragmentation or picnotic nuclei) related to induced
cell death/apoptosis. In parallel, DNA from T24 cells
was stained with propidium iodide at different time
points after infection to analyze cell cycle by using
flowcytometry.Inallexperiments,untreatedT24cells
and cells treated with staurosporine and camptothecin
(apoptosis inducers) were included as negative and
positive controls, respectively.
Our results showed that neither BCG nor M. brumae
induce apoptosis to T24 bladder cells. There were a
similar percentage of early apoptotic cells between
BCG, M. brumae and non-infected cells detected by
flow cytometry. DNA fragmentation and presence
of caspase 3 in the cytoplasm were not detected
in infected cells. SEM and TEM experiments also
showed a similar percentage of apoptosis between
mycobacteria-infected and non infected T24 cells.
In all the experiments, T24 cells treated with both
apoptotic inducers were positive. When cell cycle was
analyzed, the results showed that M. brumae arrests
the cells at G0/G1 phase, whereas BCG arrests the
cells at G0/G1 and S phase.
In conclusion, M. brumae inhibits tumor growth by
leading the cells into cell cycle arrest, and does not
induce apoptosis.
P83
IN-DEPTH SURVEY OF MyCOBaCTERIUM
avIUM SUBSP. paRaTUBERCULOSIS (MAP)
GENOTYPES IN GERMANY
Petra Möbius, Isabel Fritsch, Heike Köhler
Friedrich-Loeffler-Institut, Institute of Molecular
pathogenesis, nRL for paratuberculosis, Jena,
germany
MAP is the causative agent of paratuberculosis. This
chronic enteritis affects primarily domestic and wild
ruminants. The agent is wide-spread in cattle herds
in Europe. The current study aimed at understanding
the diversity of MAP strains in Germany and to
unveil co-infections and transmission routes. Strains
typed originated from different regions, hosts, herds,
animals, and tissues.
A total of 330 MAP isolates were sourced from 300
cattle in 128 herds. Additional isolates were from 8
further species (sheep, goat, red deer, fallow deer, roe
deer,mouflon,donkey,andman).Strainsoriginated
from eleven Federal States in Germany. Fifty isolates
from two to six tissues per individual animal (n=15, 6
herds) were analysed for co-infections at the individual
animal level. Appreciating the relatively low genetic
heterogeneity of MAP, three DNA-based genotyping
methods were applied in combination: IS900-RFLP
(BstEII, PstI)-, MIRu-vNTR (8 markers)-, and SSR (3
loci) - analysis.
About 50 combined MAP genotypes were detected. A
discriminatory index of 0.97 was achieved, considered
sufficient for epidemiological studies. Most of the
isolates belonged to the main MAP-Type-II group. Only
isolates from five sheep and two cattle belonged to
the main MAP-Type-I/III group. Four common strains
weredetectedin13upto31holdingsoffiveuptonine
Federal States. A plethora of rare or single genotypes
(i.e. from man) were found. No host preference of
genotypes within MAP-Type-II group was unveiled.
The diversity of MAP within cattle herds varied. up
to six genotypes could be found in individual herds,
depending on frequency of animal trade. Detection of
different MAP genotypes in tissues of four individual
animals was interpreted as co-infection by different
strains. Within a restricted area (Eifel National Park)
with known shared habitats of wild-living and farmed
animals, genotyping results imply that specific MAP
strains had been transmitted between and within
these species. By this, red deer and cattle maintained
areservoirforspecificMAPgenotypesnotfoundin
other areas of Germany.
These results represent an unprecedented detailed
overview of MAP genotypes in Germany. Multi-target
genotyping, as applied here, may help to unveil MAP
transmission routes between animals and different
species, as well as the entry routes of the organisms
into the environment and food chain.
103
P84
STABILITY OF GENOTYPING TARGETS
IN MyCOBaCTERIUM avIUM SUBSP.
paRaTUBERCULOSIS (MAP) UPON
CULTIVATION ON DIFFERENT MEDIA, In
vITRO AND In vIvO PASSAGE, AND NATURAL
INFECTION
Nadine Kasnitz, Heike Köhler, Petra Möbius
Friedrich-Loeffler-Institut, Institute of Molecular
pathogenesis, nRL for paratuberculosis, Jena,
germany
MAP - the causative agent of paratuberculosis affects domestic and wild ruminants worldwide.
Recently, we suggested a combination of different
typing techniques to yield a sufficient discriminatory
power for exhaustive epidemiological studies.
In order to challenge the reliability of this approach the
stabilityofdefinedMAPgenotypeswasinvestigated
after: (A) sub-cultivation on six different media, (B)
12 in vitro passages, and (C) in vivo (goat) passage.
Various sub-cultures of type, reference and 24 field
strains as well as inoculation strain re-isolated from
different tissues of 11 goats were genotyped by MIRuvNTR-, SSR-, and IS900-RFLP-analysis. Results
were compared with initial genotypes.
The target sequences of MIRu-vNTR analysis (Loci
292, X3, 25, 47, 7, 32) were stable after in vitro
cultivation and passages (A+B) as well as after in vivo
passage (C). Results of SSR typing at loci 8 and 9 (trinucleotides) and at locus 1 with seven G nucleotides
were also stable. However, 10 instead of 9 G repeats
were detected at locus 2 after animal passage, possibly
because of strand slippage during chromosomal
duplication. Of note, 11 and more G repeats at SSR
loci 1 and 2 could only be inconsistently analyzed due
to strand slippage during PCR, sequence reactions or
chromosomal duplication. This frequently resulted in
mixed-sequences or fading off of the base calls. We
therefore strongly recommend applying 11 as cut-off
value for SSR typing of MAP.
After in vitro cultivation (A+B) but not after a single
animal passage (C) changes in BstEII-patterns were
detected by IS900-RFLP-typing for 3 out of 23 strains
(including ATCC 19698). Rare IS900-RFLP-patterns
were affected, independent of the used medium.
Two field strains exhibited changed IS900-RFLP as
well as SSR-profiles (at stable SSR loci 8 and 9),
suggesting primary mix-isolates and selective subcultivation. Multiple isolates from individual animals
or an individual cattle herd naturally infected by MAP
showed identical IS900-RFLP- and MIRu-vNTRgenotypes, but different numbers of G repeats at
SSR locus 2. This implies strand slippage during
chromosomal duplication of bacteria in the course of
spread of infection within individual hosts and herds.
Consequently, SSR locus 2 should not be considered
as genome marker for epidemiological studies.
When keeping in mind above-mentioned limitations
104
for RFLP- and SSR-genotyping, the investigated
techniques are reliably capable of differentiating MAP
strains.
P122
PREVALENCE OF MyCOBaCTERIUM avIUM
SUBSPECIES paRaTUBERCULOSIS AND
MyCOBaCTERIUM avIUM SUBSPECIES
hOMInISSUSIS IN BIOGAS PLANTS IN THE
CZECH REPUBLIC
Monika Moravkova, Radka Pribylova, Iva Slana
veterinary Research Institute, Brno, Czech
Republic
Mycobacterium avium subspecies paratuberculosis
(MAP) is a causal agent of chronic intestinal disease
of ruminants, which induces significant economic
losses for farmers. The importance of this disease is
increased with the possible link of MAP with Crohn´s
disease in humans. Infected animals usually spread
MAP through their faeces to the environment, which
serves as source of infection for other animals.
Mycobacterium avium subspecies hominissusis (MAH)
is widespread in the nature regardless in some cases
may affect the humans (usually immunocopromised)
and animals as well. The aim of the current study
was to determine the prevalence of MAP and MAH in
the fermentors of 18 farm-scale biogas plants in the
Czech Republic using the real time quantitative PCR
(qPCR). All tested biogas plants were supplemented
by the farm excrements and fermented products were
used for fertilization or bedding. Each biogas plant
was examined at two times. Totally, MAP DNA was
detected in 44 % (8/18) of biogas plants. The number
of MAP cell in positive samples ranged around 102 –
103 per gram. MAH DNA was detected in all examined
samples and the number of MAH ranged around 103
– 106 per gram.
This work was supported by Grant No.
MZE0002716202 of the Ministry of Agriculture of the
Czech Republic and the Ministry of Education, youth
and Sports of the Czech Republic (Grant “Admirevet”
No. CZ 1.05/2.1.00/01.0006-ED0006/01/01).
P129
MOLECULAR ANALYSIS OF HUMAN ISOLATES
BELONGING TO MyCOBaCTERIUM avIUM
COMPLEx COLLECTED DURING THE YEARS
2005-2010 IN THE CZECH REPUBLIC
Michal Slany1, Vit Ulman2, Eva Kalakayova2, Iva
Slana1
1
Republic
2
Institute of public health in Ostrava, Ostrava,
Czech Republic
Members of Mycobacterium avium complex (MAC)
are widespread in the environment, and have been
isolated from many avian and mammalian species
including man. The MAC includes two principal species
M. avium subsp. avium (MAA) and M. a. hominissuis
(MAH) frequently associated with human diseases.
Avian mycobacteriosis is becoming disseminated for
vulnerable populations (patients with HIv or chronic
diseases). The aim of the present study was to state
distribution of the insertion sequence IS901 (MAA
specificlocus)inmycobacterialstrainsisolatedfrom
human patients in Moravian region identified as
members of MAC by GenProbe.
A total of 242 MAC isolates (124 patients) collected
during years 2005-2010 were put to detailed analyses.
Sputum samples were homogenized, decontaminated
with sodium laurylsulfate, and cultured on LöwensteinJensen medium. DNA isolated from single bacterial
colony was subjected to triplex real time PCR based
on insertion sequences IS901 and IS1245 according
to the previously described method by Slana et al.
(2008). Simultaneous sequence analysis (16S rRna,
ITS and hsp65) of IS901 and IS1245 negative isolates
was carried out.
The main sources of MAA infection are considered
domesticated bird, while MAH is mainly acquired from
soil. The low prevalence of IS901 (7 %) was expected,
because only few patients included in the study
reported close contact with birds (pigeons or hens).
Other mycobacteria detected within analyzed group
(124 patients) were: MAH (69%), M. intracellulare
(18%) and newly described MAC members (6%;
M. arosiense, M. chimaera, M. colombiense,
M. marseillense).
MAC members are second common cause of
pulmonary mycobacterioses after M. tuberculosis.
The most frequent MAC causing pulmonary infections
was MAH. This fact was in accordance with published
data. Low prevalence of MAA could be explained by
only occasional contact of humans with infected birds,
while exposition to contaminated soil or aerosols with
MAH is more frequent. Presented triplex real time
PCR is able to differentiate two etiological agents
(MAH, MAA) with different place of origin.
This work was supported by Grant No. MZE0002716202
from the Ministry of Agriculture and Grant “Admirevet”
No. CZ 1.05/2.1.00/01.0006-ED0006/01/01 from the
Ministry of Education, youth and Sports of the Czech
Republic.
P130
SOIL AND PLANT CONTAMINATION WITH M.
a. paRaTUBERCULOSIS FROM TISSUES OF
INFECTED ANIMALS
Marija Kaevska1, Samuel Lvoncik2, Jiri Lamka3,
Iva Slana1, Ivo Pavlik1
1
Republic
Institute of plant Biology, agronomical Faculty,
Mendel University, Brno, Czech Republic
3
Faculty of pharmacy in hradec Kralove, Charles
University in prague, Czech Republic
2
Mycobacterium avium subsp. paratuberculosis
(M. a. paratuberculosis) is a causal agent of
paratuberculosis also called Johne´s disease, an
incurablechronicinflammationindomesticandfree
living ruminants. Animals acquire the infectious agent
through ingestion, thus contaminated food and water
are most important sources of infection. Infected
animals shed high amount of M. a. paratuberculosis
in the environment, where it can survive and remain
virulent for a long time.
The aims of this study were to describe spatial
contamination of the environment on a mouflon
pasture; as well to assess the contamination of
grass and roots after surface contamination and in
depth contamination with buried tissues from animals
infected with M. a. paratuberculosis. Different organs
from deceased infected animals were laid on the
pasture on spots which were fenced to preclude
movement of animals. Also, two animals were
buried at 60 cm of depth in order to study possible
contamination of roots and plants after their decay.
After one year, samples of soil, roots and plants
were collected and examined from different locations
inside the mouflon pasture, and one control sample
site was chosen outside the area where the animals
are living. M. a. paratuberculosis DNA was present
in all of the examined sites and was more often
detected in roots than in soil. DNA was detected up
to 80 cm of depth and was spatially more widespread
than the initial hypothesis of M. a. paratuberculosis
leeching vertically in deeper layers of soil. This study
broadens our knowledge of spread and persistence of
M. a. paratuberculosis in an environment with highly
infected animals.
Acknowledgements
The work was supported by grants from the Ministry
of Agriculture (No. MZe0002716202) and the Ministry
of Education, youth and Sports (Admirevet, CZ
1.05/2.1.00/01.0006; ED0006/01/01.) of the Czech
Republic.
P134
MONITORING OF MyCOBaCTERIUM avIUM
SUBSP. paRaTUBERCULOSIS SURVIVAL IN
FERMENTED MILK PRODUCTS BY MEANS OF
CULTURE AND qUANTITATIVE REAL TIME PCR
METHODS
Barbora Klanicova1, Iva Slana1, Petr Roubal2, Petr
Kralik1
1
Republic
2
Dairy Research Institute, prague, Czech
Republic
105
Mycobacterium avium paratuberculosis (MAP),
increasingly discussed as a possible mediator
of human Crohn’s disease, is causative agent
of paratuberculosis in ruminants. It is capable to
survive diverse conditions like high temperature
(pasteurization), low temperature (refrigerated
storage) or very low pH (stomach). Infant powder
milk, cheese, cream and other milk and dairy products
might thus be considered as possible sources of MAP
for humans.
The aim of our study was to examine the survival of two
MAPfieldisolatesduringfermentationofthreedifferent
fermented milk products (yoghurt, acidophilus milk
and kefir) under laboratory conditions. Pasteurized
MAP-free milk was artificially contaminated with
106 MAP cells/ml and survival and absolute numbers
of MAP were monitored during fermentation (4 or 16
h) and after six weeks of storage at 4 °C by culture
and quantitative real time PCR (qPCR). viability of
MAP was determined by culture using Herrold’s egg
yolk medium and Middlebrook 7H10 with antibiotics,
supplemented with Mycobactin J and incubated at
37 °C for up to 12 weeks. The absolute numbers
of MAP were quantified by previously published
qPCR assays targeting F57 and IS900 loci in MAP
genome.
We confirmed that MAP can survive pH reduction;
however, longer exposure to pH below 4 in fermented
milk products seems to be crucial because it inhibits
the growth. Therefore we concluded that probiotic
cultures that can decrease pH below 4 during
fermentation could provide better inactivation of MAP
in fermented milk products.
The work was supported by grants from the Ministry of
Agriculture (No. MZe0002716202, qH81065) and the
Ministry of Education, youth and Sports (Admirevet
CZ 1.05/2.1.00/01.0006, ED0006/01/01) of the Czech
Republic.
P145
MOLECULAR ANALYSIS OF MyCOBaCTERIUM
ChELOnaE-M. aBSCESSUS GROUP
ISOLATES WITHOUT CONCLUSIVE SPECIES
IDENTIFICATION
Christiane Lourenco Nogueira1, Cristianne
Kayoko Matsumoto1, Luciano Antonio
Digiampietri2, João Carlos Setubal3, Sylvia
Cardoso Leão1
1
Departamento de Microbiologia, Imunologia
e parasitologia – Universidade Federal de São
paulo, Brazil
2
Escola de artes, Ciências e humanidades –
Universidade de São paulo, Brazil
3
Departamento de Bioquímica, Instituto de
Química – Universidade de São paulo, Brazil
nontuberculous mycobacteria are ubiquitous
environmental organisms and several species can
106
cause opportunistic infections in humans, in particular
the members of the M. chelonae-M. abscessus
group. until recently, this group was composed by
M. chelonae, M. abscessus, M. immunogenum,
M. bolletii, M. massiliense and M. salmoniphilum.
Taxonomic changes were proposed, among them
the inclusion of M. franklinii and the unification of
M. abscessus, M. massiliense and M. bolletii in a
single species (M. abscessus) with two subspecies
(M. abscessus subsp. abscessus and M. abscessus
subsp. bolletii). We analyzed a group of 28 isolates,
identified by the INNO-LiPA test as M. chelonae
CHII, with inconclusive species classification. PCRrestriction enzyme analyses (PRA) of the hsp65 gene
and ITS (16S-23S Internal Transcribed Spacer) and
DNA sequencing of 16S rDNA, hsp65, rpoB and ITS
were used for molecular characterization of these
isolates. The obtained sequences were compared
to sequences of the type strains of M. abscessus,
M. chelonae, M. immunogenum, M. massiliense, M.
bolletii, M. salmoniphilum and M. franklinii deposited
in the GenBank. Sixteen isolates were identified as
M. chelonae by PRA-hsp65 and PRA-ITS and this
identification was confirmed by DNA sequencing.
Seven isolates were identified as M. chelonae by
PRA-hsp65 and as M. abscessus by PRA-ITS.
Among these, three isolates showed highest similarity
of sequences with the sequences of M. franklinii or M.
chelonae and four with sequences of M. salmoniphilum
or M. franklinii. Three isolates showed a novel PRAhsp65 pattern and M. chelonae pattern by PRA-ITS.
Sequences were highly similar to sequences of M.
salmoniphilum or M. franklinii. The last two isolates
were identified as M. immunogenum by PRAhsp65 and M. abscessus by PRA-ITS. Sequences
were more similar to sequences of M. franklinii. In
conclusion, the identification of 12 isolates (42.8%)
was not reached by PRA plus sequence analysis and
additional molecular and phenotypic analyses are
necessary to determine the taxonomic classification
ofthesesisolates.Phylogenetictreesconfirmedthat
the INNO-LiPA® M. chelonae CHII cluster isolates
have high internal variability.
P150
CRITICAL ROLE OF TOLL-LIKE RECEPTOR
2 IN MyCOBaCTERIUM BRUMaE-INDUCED
ANTITUMOR ACTIVITY
Silvia Secanella-Fandos1, Carmen Fernández2,
Esther Julián1
1
2
Stockholm University, Stockholm, Sweden
Mycobacterium bovis BCG is the standard treatment
forsuperficialbladdercancer.Untilnow,theprecise
mechanisms by which BCG acts as antitumor agent
are not fully understood. But it is widely recognized
that mycobacteria are a source of modulating
molecules of the human immune system, and some
of these antigens are recognized by Toll-like receptors
(TLR), which are directly involved in the antitumor
immunity. In this sense, a role of TLR2 and TLR4 in
cell activation mediated by BCG has been described,
and some previous studies suggest a significant
function of TLR receptors on BCG antitumor activity.
However, nothing is known about TLR function on
antitumor activity of M. brumae, a new proposed
agent for immunotherapeutic treatment.
To identify a possible role of TLR signaling, the
cytotoxicity against tumor cells of M. brumae-activated
bone marrow macrophages (BMM) derived from TLRdeficientmicewasstudied.Firstly,wemeasuredthe
antitumor activity of M. brumae, BCG and M. gastri
(as negative control) against a murine tumor bladder
cell line (MB49). Secondly, BMM from C57/BL6 wild
type, TLR-2, TLR-4 and MyD88-knockout mice were
infected with different mycobateria, and cytokine and
chemokine production was detected. Finally, the
cytotoxic activity against tumor cell of mycobacteriaactivated BMM was analyzed.
Our results showed that both BCG and M. brumae
directly inhibit murine tumor cell proliferation. In
addition, BCG and M. brumae infection trigger
a significantly higher cytokines and chemokines
production in BMM, in comparison to cytokineproduction induced by M. gastri or non-infected cells. In
all cases, the production of cytokines and chemokines
is markedly TLR-2 and MyD88-dependent, and only
in part depends on TLR-4 signaling. Strikingly, M.
brumae elicit a higher release than BCG of IL-12 and
IP-10, two crucial molecules in bladder antitumor
therapy. Finally, BCG and M. brumae-activated BMM
showed cytotoxic activity dependent on TLR-2 and
MyD88, but not TLR-4.
Overall,ourfindingssuggestthatBCGandM. brumae
antitumor activity depends on TLR-2 and MyD88
signaling, while TLR-4 4 is only partially required for
the antitumor activity of mycobacteria.
P165
MYCOBACTERIAL INFECTIONS IN WILDLIFE:
FIRST REVIEW FOR SLOVENIA
Mateja Pate, Urska Zajc, Darja Kusar, Diana Zele,
Gorazd Vengust, Tina Pirs, Matjaz Ocepek
University of Ljubljana, veterinary Faculty,
Ljubljana, Slovenia
Wildlife is recognised as an important reservoir of
mycobacterial infections that seriously endanger the
efforts to control and eradicate bovine tuberculosis
(BTB) in countries with persistent BTB. Since 2009,
SloveniahasbeenofficiallyfreeofBTB,buthasnot
yet monitored for the presence of mycobacteria in wild
animal species. Therefore, the aim of this work was
to asses the prevalence of mycobacterial infections
in wildlife.
A total of 321 apparently healthy free-range wild
animals were investigated: red deer (n = 122), roe
deer (n = 84), wild boar (n = 43), fallow deer (n = 25),
wolf (n = 19), badger (n = 8), chamois (n = 8), fox (n =
6),mouflon(n=2),polecat(n=1),ibex(n=1),stone
marten (n = 1), and jackal (n = 1). Samples of liver and
mandibular, mediastinal and mesenteric lymph nodes
were Ziehl-Neelsen stained and cultivated using
Stonebrink, Middlebrook 7H10, Löwenstein-Jensen
with pyruvate, Löwenstein-Jensen with glycerine and
MGITmedia.Cultureswereidentifiedonthebasisof
colony morphology, GenoType Mycobacterium CM
and AS assays (Hain Lifescience), and 16S rRNA
gene sequencing. Mycobacteria were isolated from
39/321 (12.15%) animals, namely from red deer
(17/122), roe deer (8/84), wild boar (7/43), fallow deer
(6/25) and jackal (1/1). The most frequently detected
species was M. peregrinum (n = 8), followed by M.
avium (n = 7), M. intracellulare (n = 4), M. engbaekii
(n = 4), M. confluentis (n = 2), M. fortuitum (n = 2)
and M. terrae(n=2).Singleisolateswereidentified
as M. celatum, M. neoaurum, M. nonchromogenicum
and M. vaccae,andsixcouldonlybeidentifiedtothe
genus level. Members of Mycobacterium tuberculosis
complex were not detected.
Thisworkprovidedthefirstinsightintothesituation
regarding mycobacterial infections in wild animals
in Slovenia. With the exception of M. avium, most
of the discovered mycobacterial species could not
be regarded as highly hazardous for the spill-over
into domestic livestock or human population as they
represent conditionally pathogenic or non-pathogenic
mycobacteria.
P166
GENOTYPE ASSAY COUPLED WITH 16S RRNA
AND RPOB SEqUENCING: A GOOD APPROACH
TOWARDS IMPROVED IDENTIFICATION OF
NONTUBERCULOUS MYCOBACTERIA IN A
VETERINARY LABORATORY?
Mateja Pate1, Darja Kusar1, Urska Zajc1, James
Higgins2, Patrick Camp2, David Farrell2, Doris
Bravo2, Tod Stuber2, Matjaz Ocepek1
1
University of Ljubljana, veterinary Faculty,
Ljubljana, Slovenia
2
national veterinary Services Laboratories,
ames, Iowa, USa
Discoveries of novel species of mycobacteria can
complicate efforts to properly assign accurate and
up-to-date taxonomic information to the isolates.
Conventional methods have mostly been replaced
with molecular assays, but no gold standard for
identificationofmycobacteriaisavailable.Inthescope
of a survey on mycobacteria from various animal
species, a pilot study was conducted to improve
identification of nontuberculous mycobacteria in a
107
veterinary laboratory.
A panel of 49 isolates was used to compare GenoType
(GT) Mycobacterium CM/AS assays and sequencing
of 16S rRNA and rpoB genes. Isolates originated
from33aquariumfishand16wildanimals(reddeer,
roe deer, wild boar and fallow deer). Assignation
of isolates to species was performed using BLAST
analysis of rpoB and 16S rRNA sequence depositions
in the NCBI and RIDOM web-servers. Isolates were
identifiedasM. celatum (n=1), M. cheloane (n=3), M.
confluentis (n=1), M. engbaekii (n=3), M. fortuitum
(n=5), M. gordonae (n=1), M. intracellulare (n=2),
M. kansasii/M. gastri (n=2), M. marinum/M. ulcerans
(n=9), M. neoaurum (n=1), M. nonchromogenicum
(n=1), M. peregrinum (n=5) and M. terrae (n=5). Ten
isolateswereidentifiedtothegenuslevelonly.
A complete concordance of the results generated
by all three methods was observed for 13 isolates
belonging to M. marinum/M. ulcerans (n=8), M.
celatum (n=1), M. gordonae (n=1), M. fortuitum (n=1)
and M. chelonae (n=2). However, incongruence
of the results of all three methods was observed
in 4 cases. Comparison of the sequencing results
was not possible for 5 isolates due to poor-quality
sequences. For 3 isolates, discrepancy between GT
and sequencing results was detected. 16S rRNA and
rpoB sequencing results gave conflicting species
assignmentsfor9isolates.Thiscouldreflectdifficulties
when identifying closely related species, deposition
of poor-quality or misidentified sequences in public
databases, lack of a comprehensive database for
rpoB sequences, uncertainty about expected intra/
inter-species sequence variability and possibility of
lateral transfer of rpoB gene.
In conclusion, a combination of colony morphology, GT
assay and 16S rRNA or rpoB sequencing proved useful
for resolution of mycobacterial species encountered in
this study. However, in case of incongruent results or
species not supporting differentiation by sequencing,
conventional phenotypic methods or PCR assays
targeting the species-specific genes should also be
considered.
P167
NON-PATHOGENIC MYCOBACTERIUM FOR
THE TREATMENT OF NON-INVASIVE BLADDER
CANCER
Silvia Secanella-Fandos1, Estela NogueraOrtega2, Hasier Eraña2, Jofre Gasión2, Marina
Luquin1, Esther Julián1
1
2
Spain
Bladder cancer is the most common malignant
disease of the urinary tract. Approximately, 75-85 %
ofpatientswithbladdercancerhavediseaseconfined
108
to the mucosa and submucosa when it is diagnosed.
After transurethral resection, intravesical instillation
ofliveBCGiscarriedout.DespitetheBCGbenefits
in preventing tumor recurrence and progression, this
treatment has limitations and causes some problems
that compromise its use. Apart from not severe
toxicity associated problems, some patients suffer
more serious side effects, even including systemic
BCG infection. Because of these drawbacks it is
necessarytofindalternativestotreatbladdercancer,
more efficient and safer than live BCG. These
facts, lead us to search new tumor immunotherapy
agents,inparticularweaimedtofindnon-pathogenic
mycobacteria with antitumor activity.
After choosing a group of non-pathogenic
mycobacteria, we assessed their direct antitumoral
effect on grade 1 (RT4, SW780 and MG-Hu-3),
grade 2 (RT112 and 5637) and grade 3 (T24 and J82)
human bladder cancer cell lines. We studied both the
mycobacteria ability to inhibit tumor proliferation and
to trigger cytokines production. Therefore, we selected
themostefficaciousmycobacteriaasantitumoragent
and analyzed their capacity to activate an immune
response crucial for an indirect antitumor activity.
Cytokine production and activation expression
markers (CD40, CD80 and CD86) were detected on
mycobacteria infected-J774 murine macrophages.
Moreover cytokine production and cytotoxic activity
against tumor bladder cells of mycobacteria-infected
peripheral blood cells were analyzed.
Our results showed that the majority of nonpathogenic mycobacteria are unable to inhibit tumor
growth. However, among all mycobacteria studied, M.
brumae shows increased cytotoxic activity than BCG
on grade 1 and 2 tumor bladder cells, and a similar
cytotoxic activity than BCG against grade 3 cells. M.
brumae also triggers the production of cytokines in
infected bladder cells. In addition, this mycobacterium
induces the expression of activation markers and
the production of pro-inflammatory cytokines on
infected murine macrophages and PBMC. Finally, we
demonstrated that M. brumae-activated PBMC show
cytotoxic activity against T24 bladder cancer cells.
In conclusion, our results suggest that M. brumae
could be a possible alternative to BCG on bladder
cancer treatment.
P168
SPECIFIC FORMULATION OF MYCOBACTERIA
IMPROVES ITS ANTITUMOR ACTIVITY AGAINST
BLADDER CANCER CELLS
Estela Noguera-Ortega1, Núria Blanco-Cabra2,
Mónica Roldán3, Marina Luquin2, Esther Julián2
1
Spain
2
3
Servei de Microscopia. Universitat autònoma de
Mycobacterium bovis bacillus Calmette-Guerin
(BCG) is currently the most effective agent for
therapy of intermediate and high risk superficial
bladder cancer. Following transurethral resection, live
BCG is administered in order to prevent recurrence,
tumor progression and prolong survival. Although
it has been used for more than thirty years, little is
known about the interaction between mycobacteria
and bladder cancer cells. For instance, considering
that mycobacteria have a highly hydrophobic cell wall
which makes them form clumps, it is understandable
that aggregation can influence in the efficacy of
mycobacteria in inhibiting tumor growth.
In order to deeper into this topic, in the present work
weaimedtostudytheefficacyofdifferentformulations
on disaggregating mycobacteria clumps and on
inhibiting tumor growth.
We prepared different formulations of BCG
and Mycobacterium brumae, a non-pathogenic
mycobacterium which has a similar effect to BCG as
antitumor agent. Clumping and mycobacteria viability
were analyzed for each formulation. After selecting
the most innocuous preparations, T24 human and
MB49 murine bladder cancer cell lines were infected
with formulated mycobacteria and tumor growth
inhibition and IL-6 and IL-8 cytokine production were
measured.
Our results indicate that both BCG and M. brumae
preparedusingsomeformulationsreduceefficiently
tumor cell proliferation. Moreover, formulated
mycobacteria trigger higher production of cytokines in
bladder tumor cell cultures than mycobacteria prepared
as it is currently used in patients’ treatment.
We can conclude that BCG and M. brumae in this
kind of formulation could be a suitable alternative to
the present BCG treatment for non-invasive bladder
cancer.
P191
PULMONARY TUBERCULOSIS CAUSED BY M.
SzULgaI: CASE REPORT
Kemal Tekin1, Ferhat Onur Ural2, Mustafa Guney1,
Seyfettın Gumus2, Ali Albay3
1
Department of Medical Microbiology, gulhane
Military Medical academy, ankara, Turkey
2
Department of pulmonary Medicine, gulhane
Military Medical academy, ankara, Turkey
Pulmonary tuberculosis is a contagious and life
threatening disease, still continues to be a public
health problem in all countries. Generally caused
by M. tuberculosis complex but nontuberculosis
mycobacteria, especially M szulgai is less causative
agent.
Inthiscasereportwepresentthediagnosisofafiftyfour years old man who has pulmonary tuberculosis
caused by M. szulgai. The patient admitted our
hospital with complaining of cough with hemorrhagic
sputum, weakness and weight loss. In addition to
these symptoms he has an anamnesis of pulmonary
tuberculosis caused by M. tuberculosis twenty years
ago.Subsequenttoconsolidationandinfiltrationareas
were observed by chest X-ray and computerized
tomography;gastricfluidexaminationwasperformed
comprising acid fast staining (2+ positive), PCR
and culture. According to all the diagnostic tests,
pulmonary tuberculosis caused by M. szulgai was
reported and antituberculous therapy (isoniasid,
rifampin, pirazinamid and etambutol) was initiated.
M. szulgai is a scotochromogenic species (at 37 0C) that
causes pulmonary disease resembling M. tuberculosis
infections and extrapulmonary infections (olecranon
bursitis, cutaneous infection, osteomyelitis etc.). In
addition to this clinic manifestation, disseminated
infection occurs in immunocompromised patients.
First-line antituberculosis drugs can be used for the
treatment of M.szulgai infections.
By this case report, we call attention to the nontuberculosis mycobacteria that may also be
considered as a cause of pulmonary tuberculosis,
especially when isolated from patients with
symptoms.
P195
BLOODSTREAM INFECTION BY
MyCOBaCTERIUM aBSCESSUS: A CASE
REPORT
Serhat Duyan1, Mustafa Guney1, Erman Ates2,
Abdullah Kilic1, Ali Albay1
1
gulhane Military Medical academy, School of
Medicine, Department of Medical Microbiology,
ankara, Turkey
2
gulhane Military Medical academy, School of
Medicine, Department of pediatrics, ankara,
Turkey
10-year-old female patient was diagnosed with stage Iv
neuroblastoma, followed in department of pediatric at
Gulhane Military Medical Academy Training Hospital,
Ankara. Bone marrow transplantation was done. It was
considered that transplanted tissue was engraftment
after the 28th day of tranplantation. She had a seizure
with shaking fever which was 400C, after the 118th
day of transplantation. Ralstonia spp. was grown on
her blood culture. Appropriate antibiotic treatment was
started immediately and her clinical appearance was
improved instantly. The last blood culture which was
taken before her discharge was detected as positive.
Small, white colonies were seen in blood agar media,
which was isolated from positive blood culture bottles
within 48 hours. Identification of this bacteria was
made by MALDI-TOF MS (Matrix-assisted laser
desorption/ionization mass spectrometry). It was
identified as Mycobacterium abscessus. Acid fast
stain was made to confirm this result. Acid-fast
bacili were seen on microscobic examination. PCR
109
(polymerase chain reaction) was used to verify the
results. Mycobacterium abscessus was detected
by PCR. Growth of Mycobacterium abscessus was
reported to the clinic but it was also stated that it
could be a contamination. Although the possibility of
contamination, macrolid treatment was given to this
patient because of her immunosuppressive status.
She was discharged with oral antibiotic treatment.
By this case report, we call attention to the nontuberculosis mycobacteria (M. abscessus) when
isolated from immunosuppressive patients. New
systems like MALDI-TOF MS are useful for early
detection of this kind of rare microorganisms.
P203
PULMONARY DISEASE BY NONTUBERCULOUS
MYCOBACTERIA . A 10 YEARS STUDY
Maria Teresa Tortola Fernandez 1, Carmen
Aleman2, Meritxell Espuga3, Israel Molina4,
Jose Angel Rodrigo5, Nuria Saborit6, Asuncion
Seminario7, Teresa Soriano2, Alba Torrent8, Nuria
Martin-Casabona1
1
Microbiology Service, Barcelona, Spain
2
Internal Medicine, Barcelona, Spain
3
Unit of prevention in Labor, Barcelona, Spain
4
Infectious Diseases Service, Barcelona, Spain
5
preventive Medicine Service, Barcelona, Spain
6
Tb nurse Case Manager, Barcelona, Spain
7
pneumology Service, Barcelona, Spain
8
pediatric pneumology Service, Barcelona, Spain
Objective: To describe the epidemiological
characteristics, etiology, predisposing factors,
treatment and outcome of lung disease by
nontuberculous mycobacteria (NTM) diagnosed in
our hospital during the years 2000-2010.
Material and Methods: We reviewed the medical
records of patients diagnosed with NTM lung infection
between 2000 and 2010 in our hospital. We collected
epidemiological,
microbiological,
predisposing
factors, treatment and evolution data. For diagnosis
of pulmonary infection we followed the criteria of
the American Thoracic Society. All specimens were
digested and decontaminated using the method
described by Kent and Kubica. After decontamination
and concentration, the specimens were prepared
for: 1/ microscopic examination; 2/ samples were
inoculated in MGIT medium and incubated in Bactec
MGIT 960 (Becton Dickinson Diagnostic Instrument
System. Maryland, USA). Identification was
performed with GenoType® Mycobacterium CM/AS
(Hain Lifescience GmbH).
Results: Of the 172 patients enrolled, 54 (31.3%)
met clinical criteria for NTM lung disease. They were
30 men and 24 women with a mean age of 56.93
± 17.69 years (range 14-91). Thirty-one (57.4%)
patients showed respiratory predisposing factors,
the most frequent was bronchiectasis 12 (38.7%).
110
NTM species isolated from patients with pulmonary
disease were: 16 M. avium complex (13 M. avium, 3
M. intracellulare), 11 M. kansasii, 9 M. scrofulaceum,
6 M. xenopi, 2 M. abscessus, 2 M. mucogenicum,
2 M. celatum, 1 M. chelonae, 1 M. fortuitum, 1 M.
gordonae, 1 M. malmoense, 1 M. noncromogenicum
and 1 M. simiae. Thirty-four (62.9%) patients were
treated, 5 patients were not treated and 15 patients
were not recorded. Regarding the evolution of patients
treated, 19 (55.8) cases had no record of evolution but
we had it in 15 (44.1%). Those patients treated and
with known evolution (15), 13 (86.6%) were cured, 1
(6.6%) remained in treatment at the time of the study
and 1 (6.6%) died of causes unrelated to infection
with NTM. The most common antibiotic combination
(5) was rifampicin, ethambutol and clarithromycin.
Conclusions: 1/ One-third of the patients met clinical
criteria of NTM pulmonary disease. 2/ M. avium
complex, M. kansasii and M. scrofulaceum have been
the species most frequently associated with lung
disease in our hospital. 3/ Thirteen (86.6%) of the
treated patients and known evolution were cured.
P213
CASE REPORT OF MyCOBaCTERIUM
TILBURgII INFECTION
Timur S. Akpinar1, O.K. Bakkaloglu1, Burak Ince1,
O. Kaya Koksalan2, Nesimi Buyukbabani3, Bulent
Saka1, Nilgun Erten1, Zeki Kilicaslan4, Cemil
Tascioglu1
1
Dept. of Internal Medicine, Istanbul Med.
Faculty, Istanbul Univ., Istanbul, Turkey
2
Detae, Istanbul University, Istanbul, Turkey
3
Dept. of pathology, Istanbul Med. Faculty,
Istanbul Univ., Istanbul, Turkey
4
Dept of Chest Dis. and Tuberculosis, Istanbul
Med. Faculty, Ist. Univ., Istanbul, Turkey
The advent of new mycobacterial identification
techniques resulted in an upsurge in the number of
newly described mycobacterial species, increased
detection from direct specimens, which negate the
need for culture, finally in heightened interest in
mycobacterial infections. We herein report a case
of lymphadenitis caused by Mycobacterium tilburgii
from an immune-competent adult patient in the city of
Istanbul, Turkey.
A 26-year-old woman presented with a low-grade
abdominal pain of 10 months history. Her medical
history was unremarkable except for poorly recalled
childhood pneumonia. First medical evaluation
was inconclusive. Further investigation with MRI
revealed
multiple
abdominal
conglomerating
lymphadenopathies (LAP) with partly cysticnecrotic structure. PET-CT marked FDG avidities
at supraclavicular, lower cervical, hilar, subcarinal
and paraaortocaval LAP’s with sud-max up to 9.
Excisional biopsy of right axillary and left inguinal
lymph nodes showed intrasinusoidal histiocytosis,
pericapsular chronic inflammatory changes without
any neoplastic cell infiltration. Her second inguinal
lymph node biopsy was reported as diffuse nodular
histiocytosis. On the 3rd month, new cervical LAP
and exudative ascites were detected. Ascites
assessment was inconclusive. Excisional biopsy of
new left cervical LAP showed histiocytic infiltration
with widespread intra-cytoplasmic acid-fast bacilli.
She was then initiated to HRZE regimen. under this
treatment she presented with pulmonary symptoms
andreticulonodularinfiltrationonchestCT.HerESR
and CRP concentrations were persistently high.
Because of disease progression under treatment
specimen was directed for PCR identification. M.
tilburgiiwasidentifiedbyusing16SrDNAsequencing
from FFPE-cervical and -inguinal lymph nodes.
Afterhavingreceivedtheunambigiousidentification,
the treatment was reverted to CLR, CIP, rifabutin,
EMB, AMK. At the 10th month of her treatment she
presented with chylous ascites. Thoracic duct injury
was suspected but further intervention was postponed
because of ascites complications. At the end of the
2nd year of her diagnosis, she died of sepsis due to
a secondary bacterial peritonitis attack while she was
still under antimycobacterial treatment for more than
20 months.
To our knowledge here we present the 8th case from
which M. tilburgiiismicrobiologicallyidentifiedasthe
causative agent and mark M. tilburgii as a pathogen
for severe disseminated lymphadenitis in immunecompetent patients
P216
THE DISABILITY IN LEPROSY DUE TO SOCIAL
STIGMA
Hernando Yesid Estupiñán Velásquez1, Debora
Villa Villa2, Wellman Ribon3
1
Molecular, Bucaramanga, Colombia; Master
Student Biomedical Science, Universidad
Industrial de Santander, Colombia
2
Secretaría de Salud de Santander ,
Bucaramanga, Colombia
3
Background: During 2010 leprosy has been the
cause of more than 13000 new cases with grade-2
disabilities of the 228,474 reported. As a result, World
Health Organization (WHO) implemented a program
in order to reduce about 35% the occurrence of
disability. In Colombia, disabilities cases are 27 of the
295 new cases, considering Santander department
as a locality with a high disease burden, due to
21% of cases with disability. High levels of cases
areprobablyinfluencedbytheirlocationinurbanor
rural places or by the appearance of the stigma in
the society, such as, the fear to promote isolation of
the disease and cause the emergence of stereotypes
thatsegregatetheill,inducingattitudesthatdifficultit
identificationandpromotesthedisability.Objective:
to determinate social stigma as promoter of disability.
Methods: a data collection tool was applied to 120
people selected by simple random sampling, for
establish the socio-demographic characteristics, the
knowledge state about leprosy, and the establishment
of stigmatizing attitudes. The analysis was carried out
by percents using the calculation tool “Excel” with
license present in the “uIS”. Result and discussion:
The people was analyzed and classified onA, B, C
and D groups according to their concepts about the
leprosy, establishing as evaluative judgments, the
recognition of the disease, the route of infection, the
firstsymptomsandthestateofhealing.Wedetected
that about 22% of the population know to depth the
disease and 67% says they´re not afraid of contagion,
but between 60 and 85% of them have 17 different
attitudes that evidence ill rejection. In consequence,
weidentifiedtheexistenceofgapsontheconception
of the disease. 69% of the people most knowledgeable
about leprosy expressed attitudes of isolation of the
sick and more that 65% related leprosy with leper
and misshapen stereotypes, indicating the presence
of stigma and rejection of the disease. Therefore
it was set for the first time the state of knowledge
about leprosy in Santander and it was determined
the components of the stigma that affect sick and
modifies the attitudes of the groups they frequents,
isolating and avoiding its identification, causing the
emergence of disability. Conclusions: the stigma in
Santander people produces attitudes that require a
future academic intervention to promote the objectives
of the strategy of the WHO for the identification of
the sickness and for the reduction of it disability.
Acknowledgments: Mycobacterium, Laboratorio de
Investigación y Extensión, Grupo de Inmunología y
Epidemiología Molecular, Maestría Ciencias Básicas
Biomédicas, universidad Industrial de Santander,
Colombia.
P218
PULMONARY MYCOBACTERIOSIS CAUSED BY
MyCOBaCTERIUM pEREgRInUM IN AN OLD
WOMAN
Ionela Sorina Muntean1, Adriana Drăgan1,Daniela
Homorodean2, Sven Hoffner3
1
pneumophtisiology hospital Brasov, România
2
Leon Daniello pneumophtisiology Clinical
hospital Cluj-napoca, România
3
Supranational Reference Laboratory Stockholm,
Control, Stockholm, Sweden
A 75 years old woman, never-smoker, with pulmonary
simptomatology, was referred to our hospital
111
because of wheesing, productive caugh and fever.
Low immunity status was present due to associated
diseases, like: hypertension, diabetes mellitus,
rheumatic fever, Chronic Obstructive Pulmonary
Disease. The chest X-ray and Computer Tomograph
showed nodular shadows in the upper left lobe and
no pulmonary lesions in progress. Two sputa and
one bronchoalveolar lavage had negative smears,
but only one of the three correspondent cultures
was positive. The biochemical tests revealed the
presence of a non-tuberculous mycobacteria. The
laboratory tests also showed very high values for
erythrocyte sedimentation rate (over 100 mm). The
clinician started the therapy very soon, before having
the results for species identification. Treatment
consisted of Isoniazide, Rifampicin, Pyrazinamide and
Ethambutol.Afterreceivingthespeciesidentification
result (Mycobacterium fortuitum) from the National
Reference Laboratory, the treatment was continued
with a combination ofAmikacin and Ofloxacine, for
2 months. The patient’s status improved and finally
was considered cured. The more detailed results
came from the Supranational Reference Laboratory
Stockholm and showed the presence of M. peregrinum,
a variety included in M.fortuitum group. We described
a rare case of pulmonary mycobacteriosis due to
M.peregrinum in an old woman with underlying
diseases.
Conclusion:
1. Rapidandcorrectidentificationof
Mycobacterium clinical isolated strain is
important for the tratment of the patient;
2. Clinicians should be prepared to diagnose and
treat mycobacteriosis.
P226
MOLECULAR IDENTIFICATION OF
MyCOBaCTERIUM avIUM COMPLEx (MAC)
MEMBERS RECOVERED FROM CLINICAL
SAMPLES IN A HOSPITAL IN A 3-YEAR PERIOD
Julio Alvarez1, Beatriz Romero 2, Claudio
Cuartero3, Javier Bezos2, Carmen Casal
Comendador2, N. Sánchez2, Johanna Gimeno2,
Lucas Domínguez4, Enrique Gómez-Mampaso3
1
(vISavET), Universidad
Complutense de Madrid, Madrid, Spain; Servicio
de Microbiología, hospital Ramón y Cajal,
Madrid, Spain
2
Complutense de Madrid, Madrid, Spain
3
Servicio de Microbiología, hospital Ramón y
Cajal, Madrid, Spain
4
Complutense de Madrid, Madrid, Spain;
Departamento de Sanidad animal, Facultad de
112
veterinaria, Universidad Complutense de Madrid,
Madrid, Spain
MAC infection may cause i) pulmonary disease in
immunocompetent patients that suffer from other
predisposing diseases ii) lymphadenopathies (mainly
in children), iii) generalized infections usually in
immunocompromised patients, and iv) pulmonary
disease in immunocompetent patients without
other predisposing diseases (“Lady Windermere
syndrome”) primarily caused by M. intracellulare.
However, the evaluation of the significance of MAC
isolation from clinical samples is seriously impaired
by the wide environmental distribution of certain of
its members (mainly Mycobacterium avium subsp.
hominissuis, Mah),whichleadstothefindingofMAC
in samples from asymptomatic (and nondiseased)
individuals due to either a contamination of the
sample or a transitory colonization of the host. The
objective of this study was to identify to the species/
strain level using molecular tools a representative
selection of the MAC members recovered from clinical
samples in a hospital in 2010-2012 and to determine
if an association between species/subspecies and a
higher persistence/virulence in patients exists.
71 isolates were recovered from respiratory (n=53)
and urine (n=18) samples from patients with different
medical records (recurrent infection with MAC, vIH,
predisposingrespiratorydiseases…)andidentifiedas
MAC using commercial probes. Detection of insertion
sequences IS1245/IS901 and sequencing of the hsp65
and rpoβ genes and the internal transcribed spacer
were carried out to perform species/subspecies/
strain identification. A subset of isolates was also
subjected to MALDI-TOF analyses, and all laboratory
results were then compared with the apparent clinical
significanceoftheisolates.
Overall 52 Mah, 14 M. intracellulare, 3 M. marseillense
and 2 M. chimaera isolates were identified, with
patients with chronic MAC infections usually
harboring M. intracellulare. A notable degree of
genetic heterogeneity was found with the exception
of urine and a proportion of respiratory Mah isolates,
suggesting the existence of contamination of the
sample or exposure to common sources of prevalent
environmental MAC strains. The different distribution
of MAC species among patients suggest that distinct
epidemiological patterns/colonization capacities may
exist related with the environmental setting and/or
MAC species implicated.
P228
CHARACTERIZATION OF MYCOBACTERIUM
IN WILD BIRD GROUPS DISTRIBUTED IN THE
DEPARTMENT OF SANTANDER , COLOMBIA
Jhoner Rueda1, Fernando Rondón2, Wellman
Ribon3
1
Escuela de Biología, Universidad Industrial de
Santander, Bucaramanga, Colombia
Escuela de Biologia, Universidad Industrial de
Santander, Bucaramanga, Colombia
3
2
Avian mycobacteriosis is a disease caused by different
species of mycobacteria, usually nontuberculous;
belonging to the Mycobacterium avium complex
and Mycobacterium genavense,whicharedifficultto
diagnose and control. Wild birds play an important role
in the circulation and in the ecology of mycobacteria,
having a crucial role in the spread of avian
mycobacteriosis in animal and human populations
at risk. This study aims to evaluate the presence of
mycobacterial species in wild birds in situ conditions
in rural areas of Santander Department, Colombia. In
order to reach this purpose, we took oropharyngeal
swabs and blood samples of birds, which were
analyzed by molecular techniques using PRA
method for mycobacterial species identification. At
the present, we have evaluated 500 wild birds
corresponding to thefollowing orders Passeriformes
(91.4%), Columbiformes (3.8%), Piciformes (2.4%),
Psittaciformes (1.2%), Cuculiformes (0 , 6%),
Accipitriformes (0.2%), Apodiformes (0.2%) and
Gruiformes (0.2%), showing no alteration in their
ethology at thesampling time . The results of this work
can be a good indicator to monitor and to ensure the
integrity of wild birds and the possible risk of infection
between birds and humans, in the country with
the highest diversity of birds in the world. Tracking
mycobacteria in birds should be an ongoing activity
especially in those countries that are visited by
migratory birds or those like Colombia , which are
megadiverse countries obliged to preserve these
species.
P235
MyCOBaCTERIUM aFRICanUM IN PORTUGAL:
A CASE REPORT
Luisa Sancho1, Clara Portugal1, Luisa Tancredo1,
Maria Silva1, Angela Dias1, Filomena Silva1,
Germano Sousa1
1
Laboratory of Microbiology, Department of
Clinical pathology, hospital Fernando Fonseca,
amadora, portugal
Mycobacterium africanum is rarely found in developed
countries and is most commonly found in West
African countries. It has a similar degree of infectivity
compared to M.tuberculosis but with a slower clinical
evolution.
A 6 years old child came to our Hospital with a big
supraorbital hematoma after a traumatism one month
before. She was surgical drained and the pus was
send to microbiology.
On admission, she was in good general condition and
with no fever.
Blood tests showed anemia (hemoglobin 9,8g/dl),
leukocyte count of 11 600/mm3, and platelet count
523 000/mm3.
One week later, she came again to our hospital
emergency department with supraorbital swelling with
extension to the frontal region that turns impossible
the eye opening. She was submitted to new surgical
intervention and was medicated with Amoxicilin/
clavulanic acid.
The purulent material’s first smear was negative for
acid-fast bacilli. The sample was processed by NACL/
NaOH method and cultured in both Bactec MGIT 960
(Becton-Dickison, uSA) and Lowenstein-Jensen. The
cultures became positive in 30 days and 8 weeks,
respectively.
The identification was made in BD MGIT TBc
identification test (Biomerieux) from the MGIT, with
positive result for M.tuberculosis Complex.
The M.tuberculosis Complex´s species that have
human host are M.tuberculosis, M.africanum,
M.canettii and M.bovis BCG; being M.tuberculosis
the prevalent.
Given this atypical location, it was asked a second
sample of pus. The result was confirmed with a
positive smear.
Afterthismicrobiologicalconfirmation,thepatientwas
admitted in the Pediatric ward to further study and
presumptive antituberculosis therapy with isoniazid,
rifampin. ethambutol, and pyrazinamide was started.
The positive smear morphology made us suspect other
Micobacteria different from M.tuberculosis. The culture
was send to the reference laboratory “Laboratório
de Saúde Pública Micobacterilogia / Tuberculose
– ARSLVT” and was identified as M.africanum by
Genotype MTBC (HAIN LIFESCIENCE) method.
The isolate was susceptible to classic antituberculosis
drugs.
In terms of epidemiology, this child had occasional
contacts with an uncle who lived in Senegal. We think
that is probably the source, once this country has a
high prevalence of this bacteria.
In spite of the initial presentation of this case, as a
supraorbitalabcess,thefinaldiagnosisofthispatient
was: miliar, bone and articular tuberculosis with
vertebral involvement.
P239
OCCURRENCE OF MyCOBaCTERIUM avIUM
SUBSP. paRaTUBERCULOSIS IN ROAD KILLED
WILD CARNIVORES IN PORTUGAL
Ana Matos1, Luis Figueira1, Maria Helena
Martins1, Manuel Martins1, Filipa Loureiro2, Maria
de Lurdes Pinto2, Manuela Matos3, Ana Cláudia
Coelho2
1
2
Department of veterinary Sciences, School of
agrarian and veterinary Sciences, University of
113
Trás-Os-Montes E alto Douro (Utad), veterinary
and animal Science Center (Cecav), vila Real,
portugal
3
Department of Genetics and Biotechnology;
Institute of Biotechnology and Bioengineering,
Centre of genomic and Biotechnology,
University of Trás-Os-Montes and alto Douro
(Utad), vila Real, portugal
Paratuberculosis (Johne’s disease) is a chronic
granulomatous gastroenteritis affecting ruminants
that is caused by Mycobacterium avium subsp.
paratuberculosis (Map) leading to emaciation and
death. The disease is prevalent worldwide and has a
significantfinancialimpactonthoseaffected.Cases
of Map infection have been reported previously in
free-ranging wild carnivores but there are limited data
regarding the isolation and detection of the agent in
this taxonomic order.
A survey to determine the occurrence of Mycobacterium
avium subsp. paratuberculosis (Map) was conducted
in 74 wild carnivores found death on roads in Central
Portugal from 2009 to 2012. Post mortem examination
was done and tissues were collected from wild
carnivores representing 4 families and 7 different
species. Culture was performed and acid-fast isolates
wereidentifiedbyPCRandmycobactindependency
characteristics. using a PCR-based method tissues
were screened for the presence of species-specific
IS900.
The occurrence of infected animals was 27.0%
(n=20). Infection was recorded in 6 out of the seven
studied species: 7/49 (14.3%) fox (vulpes vulpes),
3/3 (100%) beech marten (Martes foina), 2/4 (50.0%)
Otter (lutra lutra), 7/15 (46.7%) Egyptian mongoose
(Herpestes ichneumon), and 1/1 (100%) badger
(Meles meles). Map infection was not recorded in the
two genets (Genetta genetta) studied. Infection was
found in three taxonomic families: 14.3% in Canidae,
75.0% in Mustelidae and 46.7% in Herpestidae.
In total, 666 samples were studied in culture
(portions of ileocecal valve, distal jejunum and ileum,
mesenteric, mediastinal, and retropharyngeal lymph
nodes, spleen, brain, liver, lung, kidney) and 25
(3.8%) had positive results. Tissue samples were also
studied by PCR and 40 (6.0%) had positive results.
Map was grown from tissues from 8 animals. Culture
positive samples came from 4 species (fox, n=5;
beech marten, n=1; otter, n=1; Egyptian mongoose,
n=1). PCR positive samples came from 5 species
(fox, n=3; beech marten, n=2; otter, n=1; Egyptian
mongoose, n=7).
In culture, infection was less associated with spleen
and liver. In contrast, in PCR, infection was most
frequently associated with spleen and mediastinal
lymph nodes. Evidence of generalized widespread
infectionwasfoundinfive(71.4%)confirmedfoxes,
in2(66.7%)confirmedbeechmarten,in1confirmed
otter (50.0%), in 5 (71.4%) confirmed Egyptian
mongoose and in the only (100%) confirmed and
studied badger.
This study is the first to identify that Map infection
114
can be prevalent in wild carnivores in Portugal.
According to our results there was a high occurrence
of Map infection among wild carnivores and suggest
that several wild carnivores could contribute to
the persistence of Map infection within a wildlife
complex.
P240
SEROSURVEY OF MyCOBaCTERIUM avIUM
COMPLEx IN WILD BOARS IN PORTUGAL
Ana Matos1, Luis Figueira1, Maria Helena
Martins1, Manuel Martins1, Manuela Matos2, Maria
de Lurdes Pinto3, Ana Claudia Coelho3
1
2
Department of Genetics and Biotechnology;
Institute of Biotechnology and Bioengineering,
Centre of genomic and Biotechnology,
University of Trás-Os-Montes and alto Douro
(Utad), vila Real, portugal
3
Department of veterinary Sciences, School of
agrarian and veterinary Sciences, University of
Trás-Os-Montes E alto Douro (Utad), veterinary
and animal Science Center (Cecav), vila Real,
portugal
Mycobacteria belonging to the Mycobacterium avium
complex (MAC) can infect poultry, pigs and ruminants
in the food productions chains which may be a source
of food borne illnesses in humans. Mycobacteriosis
is frequently reported among wild boar populations
in Europe. The aim of this study was to assess the
epidemiological situation of MAC in Central Portugal.
Sera from 90 hunter-killed wild boars were collected
between 2009 and 2012 in natural regions of
Central Portugal and stored at -20ºC. Demographic
characteristics were recorded. Animals were
categorized in juveniles (< 8 months) and adults (> 8
months) using tooth eruption patterns.
Serological testing was carried out to all sampled
animals using a commercial indirect enzymelinked immunosorbent assay (ELISA) (ID Screen®)
test based on detection of antibodies against
Mycobacterium avium in multiple-species. Chisquared(χ2) tests were used to evaluate differences
in positivity between adults and juveniles, males and
females,presenceoflesionsandlocal.Thesignificant
level for the test was set at p= 0.05. univariate logistic
regression was used to calculate the odds ratio.
In total, 90 samples of wild boar belonging to 2 counties
in Centre-western Portugal were investigated for the
presence of antibodies against MAC. Twenty six
(28.9%) samples were positive, following the ELISA
kit manufacturer’s instructions.
The seropositivity values among males and females
were 38.5 and 61.5%, respectively (p=0,249).
Regarding age-groups, the lowest value of
seropositivity (46.2%) was found in juveniles, and the
highest (53.8%) in adults (p=0,101). Seropositivity
was high in animals with good body condition (57.7%)
but differences were not statistically significant
(p=0,992).Twenty animals did not showed lesions, 6
(30.0%) of them were seropositive. From the seventy
animals that showed any lesion consistent with
mycobacteriosis, twenty (28.6%) were seropositive.
Serologically positive animals were distributed across
the two counties and the differences were statistically
significant. Lesions consistent with mycobacteria
infection were observed in 70 (77.8%) animals.The
possible risk factors for seropositivity were evaluated.
In the univariate analysis one variable was found to be
statisticallysignificanttoseropositivity:thecounty.The
results showed an increased odds for seropositivity
when the animals belonged from Idanha-a-Nova
when compared with animals from Penamacor (OR
6.28, 95% CI 1.36, 29.09).
ELISA platforms provide inexpensive and readily
automated techniques for high number of samples.
However, availability of serum samples from wild
animals is often limited. Carcasses with various
degrees of decomposition are often the only material
available.
The results of this survey indicate that antibodies
against MAC measured by ELISA are widely
distributed in wild boars in Central Portugal.
(1), M.malmoense (1), M.xenopi (1), the type isn’t
defined (1). Critical concentrations for antibiotics
are not developed. For DST we determined MIC to
antimicrobial drugs using 96-well plates (Magellan
Bioscience,21 drugs). All species of NTM had a
different spectrum of drug susceptibility. M.avium
strains were sensitive to moxifloxacin (MIC from 2
to 4 mkg/ml),MIC for clarithomycin was 8-16 mkg/
ml. Strains of M.kansasii were sensitive to linezolid
(MIC 8-16 mkg/ml), moxifloxacin (1 mkg/ml),
clarithomycin (0.5-4 mkg/ml), rifabutin and rifampin
(1 mkg/ml). M.simiae was sensitive to moxifloxacin
only (2 mkg/ml), M.intracellulare was sensitive to high
concentrationoflinezolid(32mkg/ml),moxifloxacin(2
mkg/ml), amikacin (32 mkg/ml),clarithomycin (2 mkg/
ml). Among rapidly growth mycobacteria M.fortuitum
strains were sensitive to linezolid (4-32 mkg/ml), to
all concentrations of ciprofloxacin, moxifloxacin,
amikacin. Strains of M.chelonae and M. abscessus
were sensitive to linezolid (4-32 mkg/ml),amikacin
(16 mkg/ml),tigecycline (1-2 mkg/ml), tobramycin
(to all concentrations). Conclusion: 19 patients with
preliminary TB diagnosis were determined as patients
with mycobacteriosis. Determination of MIC to 21
antibiotics allows physicians to prescribe adequate
treatment.
P254
TB OR MYCOBACTERIOSIS: DETECTION,
SPECIES IDENTIFICATION AND DST IN
PRACTICE OF MICROBIOLOGY LAB
Tatiana Smirnova1, Elena Larionova1, Sofia
Andreevskaya1, Alena Vorobyeva1, Larisa
Chernousova1
1
Central Tb Research Institute of Rams, Moscow,
Russia
There is a problem with diagnostics of mycobacteriosis
in Russia. Species identification of mycobacterial
cultures is not performed in bacteriology labs over
Russia. Number of patients infected with NTM is
unknown. DST is not carried out for NTM. The goal
for our study was to determine the share of patients
with mycobacteriosis in the total number of TB
patients hospitalized in CTRI in the course of 20112013. Additionally MIC for NTM was performed.
1596 positive MGIT tubes were studied (from 872
patients). For suspected samples (negative results
of PCR IS6110, positive Ziehl-Neelsen stain)
species identification using GenoTypeCM/AS (HAIN
Lifescience, Germany) were carried out. Of 1596
cultures, 113 cultures (7.08%) belonged to NTM (from
75 patients). For 19 patients NTM strains belonged
to the same species were revealed from sputum
more than one time. Following spectra of NTM
strains were detected: M.avium (7), M.kansasii (4),
M.abscessus (2), M.intracellulare (2), M.gordonae
115
IGRA AND LATENT TB
P118
INDETERMINATE qUANTIFERON-TB GOLD INTUBE RESULTS IN CHILDREN: ASSOCIATION
WITH PNEUMONIA?
Giulia Lombardi1, Paola Dal Monte1, Agnese
Denicolò1, Antonella Pace1, Roberta Petrucci2,
Ilaria Corsini2, Maria Letizia Bacchi Reggiani3,
Salvatore Cazzato2, Maria Paola Landini1
1
Microbiology Unit, S. Orsola-Malpighi general
hospital, University of Bologna, Bologna, Italy
2
pediatric pneumology Unit, S. Orsola-Malpighi
general hospital, University of Bologna,
Bologna, Italy
3
Cardiology Unit, S. Orsola-Malpighi general
hospital, University of Bologna, Bologna, Italy
Introduction: Recent immunological assays based on
the measurement of released interferon-gamma after
stimulation of T-cell responses with Mycobacterium
tuberculosis specific antigens (IGRAs) have been
developed for the immunodiagnosis of latent
tuberculosis infection (LTBI). A major concern about
the use of IGRAs in children regards the possibility
of higher frequency of indeterminate results, because
their immune system can still be immature.
Aim of this study was to evaluate the frequency of
indeterminate results in children and their possible
association with diagnosis.
Methods: We retrospectively evaluated the routine
use of quantiferon-TB Gold In-Tube (qFT-IT; Cellestis,
Australia) on 445 children at the Pediatric Pneumology
unit of S. Orsola-Malpighi General Hospital during the
period April 2007-August 2012. Patients were divided
by age groups as follows: < 2 years old (n=122), 2-5
years old (n=151), 5-10 years old (n=126) and 10-17
years old (n=66).
Results: In our pediatric population: mean age was
5.4 ± 3.9 years, 246 (55%) were male, 99 (22%)
were not Italy-born, 179 (40%) were born in Italy from
immigrant families and 167 (38%) were Italian. qFTIT was positive in 70 (16%) children, negative in 353
(79%) and indeterminate in 22 (5%).
All indeterminate qFT-IT results in this study were due
to an inadequate response to PHA; 3 patients had a
known iatrogenic immunosuppression. Indeterminate
results were not statistically distributed among
different age groups (p=0.386).
Diagnosis was available in 321 (72%) children: 30
(9%) were diagnosed as LTBI, 38 (12%) as active TB,
34 (11%) as pneumonia, 219 (68%) were exposed
to a TB case. Indeterminate qFT-IT results were
distributed as follows: 0% in LTBI, 0% in active
TB, 0.5% in exposed to a TB case and 23.5% in
116
pneumonia. A statistically association with diagnosis
of pneumonia was observed (p=0.0001).
Conclusion: These preliminary data suggest that
age hasn’t effect on frequency of indeterminate qFTIT results, supporting the use of this immunological
test in young children. In our population pneumonia
seems a risk factor associated with indeterminate
qFT-IT. Immunological anergy due to pneumonia
should been further investigated.
P233
CLINICAL AND ROENTGENOLOGICAL
FEATURES IN CHILDREN WITH DIFFERENT
PROFILES OF IMMUNOLOGICAL TESTS
Anna Starshinova, Pavel Gavrilov, Natalia
Korneva, Irina Dovgaluk
St.-petersburg Research Institute of
phthisiopulmonology, St. petersburg, Russia
Objectives: to identify clinical and X-ray features in
childrenwithdifferentprofilesofimmunologicaltests
Materials and methods: At children department of
Saint-Petersburg Institute of Phthisiopulmonology
120 patients aged from 3 to 14 years were examined
during 2010-2012. All patients were assessed by
immunological tests (Diaskintest ® and quantiFERONTB Gold) and multislice computer tomography
angiography (MCT-AG). According to the results
of immunological tests patients were divided into 2
groups: I group - negative reaction of immunological
tests (n = 64), II group - with positive tests (n = 56).
Statistical analysis was performed using Statistica
8.0 and chi-square criterion.
Results: The results of measurements of
intrathoracic lymph nodes in CT angiography for the
short axis in the axial projection are presented in
Table 1.
Tabl.1 The size of the lymph nodes (mm) in children
withdifferentprofilesofimmunologicaltests
The size of the
lymph nodes
(mm)
I group (n = 64)
II group (n =56)
<0,2
46.8%(30)*
17.8% (10)
2-5
29.8% (19)
1.8% (1)
>5
23.4%(15)
80.4%* (45)
ChildrenintheIgrouphavesignificantlyhigher(46.8%)
size of the lymph nodes <0,2 mm, while in the II group
- >5 mm (80.4% vs. 23.4%, χ2=33.93, р<0,001).
Children with positive QFT-G (II) have significantly
higher intoxication syndrome in comparison with
I group (61.2% vs. 21.3%, χ2 = 17,8, p<0,001). In
73.9% cases (р<0,001) in group II calcification in
intrathoracic lymph nodes was revealed.
Conclusion: Positive results of the immunological
tests accompanied by intoxication syndrome in the
majority of cases in children are associated with the
enlargement of intrathoracic lymph nodes greater
than 5 mm and signs of specific TB inflammation.
117
AUTHOR INDEX
Abad, Yoandra 29, 101
Abadia, Edgar 22, 70
Abouljadayel, Naela 12, 19, 56
Agerberth, Birgitta 11, 18, 53
Aizikov, Dmitriy 26, 88
Ajbani, Kanchan 11, 18, 49
Akpinar, Timur S. 31, 110
Albay, Ali 27, 31, 92, 109
Aleman, Carmen 31, 110
Alende, Tatiana 23, 73
Alexandru, Sofia 11, 18, 28, 52, 95
Al-Hajoj Al-Nakhli, Sahal 12, 19, 56
Al-Hakeem, Raafat 12, 19, 56
Ali, Asho 20, 62
Alic, Amer 29, 102
Alkhovik, Olga 20, 60
Allen, Adrian 9, 17, 45
Allix-Béguec, Caroline 9, 17, 33, 44
Almeida da Silva, Pedro 20, 59
Alrabiah, Fahad 12, 19, 56
Alvarez, Julio 23, 31, 73, 74, 112
Alyapkina, Yulia 27, 93
Amaro, Ana 27, 92
Ambroggi, Martha 25, 84
Ambrosi, Alessandro 28, 99
Andersen, Åse Bengård 24, 80
Andreevskaya, Sofia 32, 115 Anthony, Richard 11, 18, 22, 26, 52, 68, 70, 88
Antonov, Victor 26, 88
Antunes, A 25, 84
Anzola, Juan Manuel 9, 16, 41, 42
Aranaz, Alicia 21, 67
Archakova, Ludmila 27, 94
Arenas-Guzmán, Roberto 29, 101
Aspindzelashvili, Rusudan 22, 70
Ates, Erman 31, 109
Augustynowicz-Kopeć, Ewa 26, 87 Azé, Jérôme 24, 78
Bablishvili, Nino 22, 70
Bacchi Reggiani, Maria Letizia 32, 116
Bachiyska, Elizabeta 10, 17, 48
Bagdasarian, Tatev 28, 97
Bakkaloglu, O.K. 31, 110
Barrera, Lucia 25, 84
Barth, Aline 10, 18, 49
Bastard, M. 24, 77
Baumanis, Viesturs 24, 76
Bauskenieks, Matiss 24, 76
Bautista, Jose Manuel 28, 98
Beckert, P. 24, 77
Bektas, Abdullah 23, 73
Bentley, Stephen 9, 16, 41
Berg, Stefan 40
Bergval, Indra 22, 68, 70
Bermingham, Mairead 9, 17, 45
118
Besirovic, Hajrudin 29, 102
Bespyatykh, J 20, 61
Bezos, Javier 21, 23, 31, 66, 74, 112
Biek, Roman 24, 77
Bimbi, Nicola 10, 17, 46
Bishop, Steve 9, 17, 45
Bjoern-Mortensen, Karen 24, 80
Blagoadeteleva, Galina 27, 91
Blanco-Cabra, Núria 31, 108
Bloemberg, Guido V. 22, 25, 27, 67, 83, 90
Boisset, Sandrine 22, 71
Boniotti, Maria Beatrice 23, 72
Bonnet, M. 24, 77
Borgdorff, Martien 22, 33, 69
Borroni, Emanuele 26, 87
Böttger, Erik C. 22, 25, 27, 67, 83, 90
Brankova, Nadia 22, 70
Bravo, Doris 30, 107
Brosch, Roland 9, 14, 35
Bryant, Josephine 12, 33
Burakova, Marina 28, 97
Bustos, Jose Ricardo 9, 16, 42
Butcher, Philip 10, 14
Buyukbabani, Nesimi 31, 110
Byun, Hyun Sub 21, 64
Bzekalava, Nino 22, 70
Cabibbe, Andrea M. 26, 86, 87
Cambau, Emmanuelle 8, 11
Camp, Patrick 30, 107
Cardoso, A 25, 84
Cardoso Leão, Sylvia 12, 19, 30, 55, 106
Carret, Gérard 22, 71
Carricajo, Anne 22, 71
Casal Comendador, Carmen 21, 23, 31, 66, 74, 112
Casali, N. 28, 96
Catanzaro, Antonino 11, 18, 28, 49, 95
Causse, Manuel 25, 81
Cazzato, Salvatore 32, 116
Celikkan, Cigdem 29, 99
Cesur, Salih 23, 27, 74, 91
Ceyhan, Ismail 23, 74
Chan Jung, Suk 21, 64
Cherednichenko, Andrey 20, 60
Chernousova, Larisa 32, 115
Chesov, Dumitru 28, 95
Chryssanthou, Erja 22, 70
Cirillo, Daniela M. 9, 26, 28, 86, 87, 99
Cittaro, Davide 9, 16, 43
Clark, Taane G 9, 10, 14, 16, 20, 36, 41, 62
Codecasa, Luigi 28, 99
Coeck, Nele 26, 86
Coelho, Ana Cláudia 32, 113, 114
Coll, Francesc 9, 16, 41
Comas, Iñaki 15, 40
Condamine, Bénédicte 9, 17, 44
Copano, María Francisca 23, 73
Corsini, Ilaria 32, 116
Coscolla, Mireia 40
Costa, Pedro 27, 92
Costa, Manuela 29, 102
Couto, Isabel 11, 18, 24, 27, 53, 79, 92
Couvin, David 22, 71
Crudu, Valeriu 11, 18, 27, 28, 49, 52, 91, 95
Cuartero, Claudio 31, 112
Cvetnic, Zeljko 21, 29, 65, 102
Cynamon, Michael 29, 102
Dadu, Andrei 11, 18, 51
Dal Monte, Paola 23, 32, 72, 116
Dara, Masoud 11, 18, 51
David, Suzana 5, 12, 25, 84
de Beer, Jessica 22, 33, 68, 69, 70
de Colombani, Pierpaolo 11, 18, 51
de Jong, Bouke 26, 86
de Juan, Lucía 21, 23, 66, 73, 74
De La Torre, Teresa Zardán Gómez 10, 17, 48
de Mendoza, Carmen 28, 98
de Vos, Margaretha 9, 16, 28, 44, 96
Deggim, Vanessa 22, 27, 67, 90
Del Portillo, Patricia 9, 10, 16, 17, 41, 42, 48
den Hertog, Alice 11, 18, 26, 52, 88
Denicolò, Agnese 32, 116
Derendorf, Hartmut 10, 18, 49
Dhole, Tapan N 25, 82
Dias, Angela 32, 113
Diel, Roland 10, 17, 47
Diez, Amalia 28, 98
Digiampietri, Luciano Antonio 30, 106
Dippenaar, Anzaan 20, 59
Dlamini, Th. 24, 77
Dogan, Gokselin 27, 92
Domente, Liliana 11, 18, 28, 52, 95
Domínguez, Lucas 21, 23, 31, 66, 73, 112
Dovgaluk, Irina 12, 19, 32, 58, 116
Duarte, EL 25, 84
Duvnjak, Sanja 21, 65
Duyan, Serhat 31, 109
Dymova, Maya 20, 60
Echemendia, Miguel 25, 82
EDCTP TB CHILD Consortium 86
Ehlers, Stefan 9, 16, 43
Eisenach, Kathleen 9
Eliseev, Platon 22, 70
Engström, Anna 10, 17, 48
Eraña, Hasier 31, 108
Erkose Genc, Gonca 29, 100
Erten, Nilgun 31, 110
Erturan, Zayre 29, 100
Erzsébet Iringó, Zaharia Kézdi 27, 93
Escamilla-Tilch, Monica 29, 101
Espuga, Meritxell 31, 110
Estrada Parra, Sergio 29, 101
Estrada-García, Iris 29, 101
Estupiñán Velásquez, Hernando Yesid 31, 111
Eszter, Vas Krisztina 27, 93
Etchart, Ana 23, 75
Fabio, Anna 20, 61
Fafutis, Mary 29, 101
Fajfar, Nataša 21, 62
Fallico, Loredana 26, 89
Farrell, David 30, 107
Fattorini, Lanfranco 11, 12, 19, 54
Fauville, Maryse 22, 68
Fernandez, Paulo 24, 78
Fernández, Carmen 30, 106
Ferraro, Giuseppina 23, 72
Ferreira, Stéphanie 9, 17, 44
Ferreira, Ana 27, 92
Ferro, Beatriz 22, 68
Feuerriegel, Silke 26, 87
Figueira, Luis 32, 113, 114
Figueroa, Maria Alvarez 28, 94
Filipenko, Maxim 20, 60
Flandrois, Jean-Pierre 22, 71
Floto, Andres 12, 19, 56
Forbicini, Giulia 20, 61
FP7 TM REST Consortium 86
Fregni Serpini, Giulia 20, 61
Fritsch, Isabel 29, 103
Gagneux, Sebastien 9, 40
Ganiats, Theodore 11, 18, 49
Gao, Qian 40
Garcia, Maria Jesus 9, 11, 16, 28, 41, 42, 98
García, Grechen 25, 29, 81, 101
García de Viedma, Darío 21, 23, 66, 73
Garfein, Richard 11, 18, 49
Garrido, Carolina 28, 98
Garzelli, Carlo 10, 17, 46
Gasión, Jofre 31, 108
Gavrilov, Pavel 32, 116
Gennari, William 20, 61
Giannoni, Federico 11, 19, 54
Gimeno, Johanna 112
Glass, Liz 9, 17, 45
Goletti, Delia 12, 15, 28, 39, 99
Gómez-Mampaso, Enrique 31, 112
Gomgnimbou, Michel 10, 17, 20, 48, 60
Gonzalez-y-Merchand, Jorge 9, 16, 41, 42
Gori, Andrea 9, 14, 35
Goria, Maria 23, 72
Gorina, Galina 22, 70
Govorun, V 20, 61
Granados, Julio 29, 101
Groessl, Erik 11, 18, 49
Grogono, Dorothy 12
Grottola, Antonella 20, 61
Gudmundsson, Gudmundur 11, 18, 53
Guducuoglu, Huseyin 23, 73
Gumus, Seyfettın 31, 109
Guney, Mustafa 27, 31, 92, 109
Gutiérrez, Alexandra 21, 66
Hasan, Rumina 20, 62
Hasan, Zahra 20, 62
Hayes, Cindy 11, 18, 49
Helguera-Repetto, Addy Cecilia 9, 16, 42
119
Henriques, V 25, 84
Hernandez, Adriana Carolina 9, 16, 42
Hernández-Neuta, Ivan 10, 17, 48
Herthnek, David 10, 17, 48
Higgins, James 30, 107
Hillemann, Doris 8
Hoffner, Sven 5, 11, 15, 18, 23, 26, 31, 37, 51, 76, 87,
88, 111
Homorodean, Daniela 11, 18, 23, 28, 31, 51, 76, 95, 111
Hubans-Pierlot, Christine 9, 17, 44
Ilina, E 20, 61
Inácio, João 27, 92
Ince, Burak 31, 110
Isaeva, Y 20, 61
Ischenko, D 20, 61
Jackson, Roberta Lynn 11, 18, 49
Jacomo, Véronique 22, 71
Jang, Yoonra 21, 64
Jang, Yun-Ho 21, 64
Jang, Jae Min 21, 64
Jansone, Inta 24, 76
Jochims, F. 24, 77
Jodal, Andreea Melinda 11, 51, 23, 28, 76, 95
Johnson, Judith 10, 18, 49
Jordao, Luisa 24, 79
Julca, Natalia 28, 98
Julián, Esther 29, 30, 31, 102, 106, 108
Kaevska, Marija 30, 105
Kalakayova, Eva 30, 104
Kamper-Jørgensen, Zaza 24, 80
Kang, Shin-Seok 21, 64
Kant, Surya 25, 82
Kao, Rowland 24, 77
Karpova, I 20, 61
Kasnitz, Nadine 30, 104
Katalinic-Jankovic, Vera 11, 21, 65
Kayoko Matsumoto, Cristianne 12, 19, 30, 55, 106
Kilic, Abdullah 31, 109
Kilicaslan, Zeki 31, 110
Kim, Narae 21, 64
Kim, Jae Myung 21, 64
Kisa, Ozgul 27, 92
Klanicova, Barbora 30, 105
Koarakozian, H. 24, 77
Kohl, Thomas 10, 17, 47
Köhler, Heike 29, 30, 103, 104
Koksalan, O. Kaya 31, 110
Korneva, Natalia 12, 19, 32, 58, 116
Korobitsyn, Alexei 26, 85
Kosloff, Barry 8
Kostryukova, E 20, 61
Krajewska, Monika 21, 22, 65, 66, 68
Kralik, Petr 30, 105
Kreiswirth, B. 28, 96
Kremer, Kristin 11, 18, 24, 51, 78
Kumar, Manoj 25, 82
Kurpina, N. 28, 96
Kusar, Darja 30, 107
120
Ladefoged, Karin 24, 80
Lamka, Jiri 30, 105
Landini, Maria Paola 32, 116
Lapenkova, Marina 27, 93
Larionova, Elena 32, 115
Larsson, Lars-Olof 22, 70
Lazarevic, Dejan 9, 16, 43
Le, Viet Hai 20, 60
Leite, CQF 25, 84
Lemus, Dihadenys 25, 82
Levterova, Viktoria 22, 70
Lidén, Ylva 26, 88
Lienhardt, Christian 11, 15, 38
Lillebaek, Troels 24, 80
Lina, Gérard 22, 71
Lipiec, Marek 21, 22, 65, 66, 68
Lombardi, Giulia 32, 116
López, Beatriz 23, 25, 75, 84
Lorenzo, Julia 29, 102
Lőrinczi, Lilla 27, 93
Loureiro, Filipa 32, 113
Lourenco Nogueira, Christiane 12, 19, 30, 55, 106
Louw, Gail Erika 9, 16, 44
Lozano, Francisco 23, 74
Lucke, Katja 25, 83
Ludannyy, Ruslan 28, 94
Luo, Tao 40
Luquin, Marina 29, 31, 102, 108
Lvoncik, Samuel 30, 105
Maaß, Silvia 9, 16, 43
Macedo, Rita 24, 79
Machado, Diana 11, 18, 24, 53, 79
Magnani, Rita 20, 61
Mairey, Mathilde 9, 17, 33, 44
Mallard, Kim 9, 16, 20, 41, 62
Mallon, Tom 24, 77
Malm, Sven 9, 16, 43
Mané, A 25, 84
Manganelli, Riccardo 26, 89
Manicheva, Olga 26, 88
Mansjö, Mikael 26, 88
Marin-Garcia, Patricia 28, 98
Markova, Nadya 28, 98
Martin, Nigel 9, 16, 41
Martin, Anandi 10, 17, 20, 25, 48, 59, 82, 83, 84
Martin, Carlos 12, 15, 38
Martin-Casabona, Nuria 31, 110
Martínez Romero, María Rosarys 25, 29, 81, 101
Martinez-Serna, Alejandra 28, 98
Martins, Maria Helena 32, 113, 114
Martins, Manuel 24, 32, 78, 113, 114
Martins Bispo, Paulo Jose 12, 19, 55
Maryandyshev, Andrey 22, 70
Mathys, Vanessa 26, 87
Matos, Ana 32, 113, 114
Matos, Manuela 32, 113, 114
Matteelli, Alberto 28, 99
Maurya, Anand Kumar 25, 82
May, Robert 10, 18, 49
Mazzola, Ester 23, 72
Mazzone, Piera 23, 72
McBride, Stewart 9, 17, 45
McCormick, Carl 24, 77
McDowell, Stanley 9, 17, 24, 45, 77
McNerney, Ruth 9, 16, 20, 41, 62
Mederos, Lilian María 25, 29, 81, 101
Memish, Ziad 12, 19, 56
Menendez, Maria Carmen 28, 98
Menting, Sandra 11, 18, 26, 52, 88
Merker, Matthias 9, 16, 28, 43, 95
Merker, M. 24, 77
Millet, Julie 23, 72
Mily, Akhirunnesa 11, 18, 53
Miotto, Paolo 9, 10, 14, 16, 26, 28, 36, 43, 87, 99
Möbius, Petra 29, 30, 103, 104
Moiseyeva, Svetlana 28, 97
Mokrousov, Igor 10, 12, 17, 19, 20, 21, 46, 55, 61, 64
Molina, Israel 31, 110
Monteserin, Johana 23, 75
Moravkova, Monika 30, 104
Morcillo, Nora 25, 83
Moya, Nuria 23, 74
Müllerová, Maria 24, 79
Muntean, Ionela Sorina 23, 76, 111
Murray, Megan 22, 69
Mustazzolu, Alessandro 11, 19, 54
Mwangoka, Grace 28, 99
Nag, Vijaya Lakshmi 25, 82
Nair, Mridul 20, 62
Nanni, Nadia 20, 61
Narvskaya, Olga 12, 19, 20, 21, 55, 61, 64
Navarro, Yurena 21, 23, 66, 73
Nebenzahl-Guimaraes, Hanna 22, 69
Nemes, Maria 27, 93
Niemann, Stefan 2, 5, 8, 9, 10, 11, 12, 14, 16, 17, 19, 24,
26, 28, 34, 40, 43, 47, 57, 77, 87, 95
Nikishova, Elena 22, 70
Nilsson, Mats 10, 17, 48
Nodieva, Anda 24, 76
Noguera-Ortega, Estela 31, 108
Norbis, Luca 28, 99
Noroc, Ecaterina 11, 18, 27, 28, 52, 91, 95
Nosova, E 20, 61
Ocepek, Matjaz 30, 107
Onur Ural, Ferhat 31, 109
Orpella-Aceret, Gemma 29, 102
Orton, Richard 24, 77
Otten, Tatiana 12, 19, 20, 21, 55, 61, 64
Ovchinnikova, Julia 12, 19, 58
Ozere, Iveta 24, 76
Pacciarini, Maria 23, 72
Pace, Antonella 32, 116
Pain, Arnab 20, 62
Paixão, E 25, 84
Palacios, Juan José 23, 73
Palomino, Juan Carlos 10, 17, 20, 25, 48, 59, 82, 83, 84
Palù, Giorgio 26, 89
Panaiotov, Stefan 10, 17, 22, 48, 70
Parkhill, Julian 9, 12, 16, 19, 40, 41, 56
Pascarella, Michela 26, 89
Pate, Mateja 30, 107
Patraulea, Mihaela 27, 93
Pavlik, Ivo 30, 105
Pavlova, Mariya 27, 94
Pecorari, Monica 20, 61
Pedersen, Matthias K. 24, 80
Peloquin, Charles 10, 18, 49
Peracchi, Marta 26, 89
Perdigao, Joao 24, 79
Petrenko, Tatyana 20, 60
Petrucci, Roberta 32, 116
Pham, Minh Chau 20, 60
Piccaro, Giovanni 11, 19, 54
Pichat, Catherine 22, 71
Pietraforte, Donatella 11, 19, 54
Pinto, Maria de Lurdes 32, 113, 114
Piro, Benoit 20, 60
Pirs, Tina 30, 107
Poggi, Susana 25, 84
Pole, Ilva 24, 76
Portaels, Françoise 12, 15
Portugal, Clara 32, 113
Portugal, Isabel 24, 79
Potapenko, Elena 12, 19, 58
Prasovic, Senad 29, 102
Preston, Mark D. 9, 16, 41
Pribylova, Radka 30, 104
Prokopenko, Anastasiya 28, 94
Puyet, Antonio 28, 98
Racic, Ivana 21, 65
Rahim, Zeaur 11, 18, 53
Ramazanzadeh, Rashid 21, 64
Ramos, Daniela 20, 59
Ramos, Jorge 11, 18, 53
Ramos-Payán, Rosalío 29, 101
Raqib, Rubhana 11, 18, 53
Rasmussen, Erik M. 24, 80
Rassu, Mario 26, 89
Rastogi, Nalin 5, 9, 22, 23, 71, 72
Refrégier, Guislaine 10, 17, 24, 48, 78
Reisberg, Steeve 20, 60
Reither, Klaus 26, 28, 86, 99
Rekha, Rokeya 11, 18, 53
Reniero, Ana 23, 75
Ribeiro, Marta 20, 59
Ribeiro, Andrezza 20, 59
Ribeiro, Luis Caiola 24, 78
Ribon, Wellman 23, 31, 75, 111, 112
Riccardi, Giovanna 8, 15, 40
Rice, L. 28, 96
Ridell, Malin 8, 22, 70
Rigouts, Leen 26, 86
Rindi, Laura 10, 17, 46
Ritacco, Viviana 22, 23, 25, 68, 75, 84
121
Ritter, Claudia 22, 25, 27, 67, 83, 90
Rodrigo, Jose Angel 31, 110
Rodrigues, Camilla 11, 18, 49
Rodríguez, Juan Germán 9, 16, 41, 42
Rodriguez-Campos, Sabrina 21, 66
Rodwell, Timothy 11, 18, 49
Roldán, Mónica 31, 108
Romancenco, Elena 11, 18, 27, 28, 49, 52, 91, 95
Romero, Beatriz 21, 23, 31, 66, 73, 74, 112
Rondón, Fernando 31, 112
Rossetti, Maria Lucia 20, 59
Roubal, Petr 30, 105
Rueda, Jhoner 31, 112
Ruesch-Gerdes, Sabine 10, 24, 28, 77, 95
Ruiz, Pilar 25, 81
Ryder, Jon 9, 17, 45
Ryoo, Soyoon 21, 64
Sabbatini, Anna Maria Teresa 20, 61
Saborit, Nuria 31, 110
Saka, Bulent 31, 110
Salazar, M. Isabel 29, 101
Salem, JI 25, 84
Samoilova, Anastasia 28, 97
Sanca, A 25, 84
Sanchez, E. 24, 77
Sánchez, N. 112
Sánchez-Chardi, Alejandro 29, 102
Sancho, Luisa 25, 32, 84, 113
Santin, Katiane 12, 19, 55
Sapozhnikova, Nadezhda 27, 94
Sardiña, Misleidis 25, 29, 81, 101
Sarker, Protim 11, 18, 53
Satana, Dilek 29, 100
Schuitema, Anja 22, 68, 70
Secanella-Fandos, Silvia 29, 30, 31, 102, 106, 108
Seersholm, Niels Jørgen 24, 80
Seminario, Asuncion 31, 110
Sengstake, Sarah 22, 26, 70, 88
Setubal, João Carlos 30, 106
Shitikov, Egor 20, 61
Shulgina, Marina 26, 89
Silva, Maria 32, 113
Silva, Carla 24, 79
Silva, Filomena 32, 113
Simonetti, Maria Tullia 23, 72
Şimşek, Hülya 23, 27, 74, 91
Singh, Amresh Kumar 25, 82
Singh Kushwaha, Ram Awadh 25, 82
Sirgel, Frick 25, 83
Skenders, Girts 5, 24, 76
Sklaney, Mary 102
Skuce, Robin 9, 17, 24, 45, 77
Slana, Iva 30, 104, 105
Slany, Michal 30, 104
Slavchev, Georgi 28, 98
Sloot, Rosa 33
Smienk, Ernst 26, 88
Smirnova, Tatiana 32, 115
122
Sola, Christophe 10, 17, 20, 22, 24, 48, 60, 70, 78
Solovyeva, Natalya 21, 26, 27, 64, 88, 94
Somoskovi, Akos 27, 90
Soriano, Teresa 31, 110
Sotgiu, Giovanni 28, 99
Sousa, Germano 32, 113
Spicic, Silvio 21, 29, 65, 102
Spies, Fernanda 20, 59
Stakenas, Petras 26, 87
Starkova, Daria 12, 19, 55
Starshinova, Anna 12, 19, 27, 32, 58, 94, 116
Strømme, Maria 10, 17, 48
Stuber, Tod 30, 107
Stupka, Elia 9, 16, 43
Supply, Philip 8, 9, 14, 17, 33, 44
Sutre, AF 25, 84
Tagliabue, Silvia 23, 72
Tagliazucchi, Sara 20, 61
Tancredo, Luisa 32, 113
Tarasova, Irina 22, 70
Tarhan, Gülnur 23, 27, 74, 91
Tascioglu, Cemil 31, 110
Tekin, Kemal 27, 31, 92, 109
Thomsen, Vibeke Østergaard 24, 80
Tizzano, Barbara 9, 12, 16, 19, 43, 57
Torrent, Alba 31, 110
Torrents, Eduard 29, 102
Torres-Carrillo, Nora M 29, 101
Tortola Fernandez, Maria Teresa 31, 110
Tortoli, Enrico 4, 5, 12
Trewby, Hannah 24, 77
Trollip, Andre 11, 18, 49
Tuin, Kiki 22, 70
Turcanu, Nadejda 11, 18, 27, 28, 52, 91, 95
Turk, Derya 29, 99
Ulman, Vit 30, 104
Uzun, Meltem 29, 100
Valente, Ilaria C. 26, 28, 87, 99
van den Boom, Martin 11, 18, 51
van Helden, Paul 9, 16, 44
van Ingen, Jakko 22, 68
van Pittius, Nico Gey 20, 59,
van Soolingen, Dick , 8, 9, 11, 14, 18, 22, 24, 33, 52, 68,
69, 70, 78
Vandamme, Peter 25, 84
Vaquero, Manuel 22, 69
Varghese, Bright 12, 19, 56
Varlamov, Dmitry 27, 93
Vasilyeva, Irina 28, 97
Vengust, Gorazd 30, 107
Victor, Thomas 9, 11, 16, 18, 44, 49
Viero, Marta 26, 89
Villa Villa, Debora 31, 111
Vishnevskiy, Boris 12, 19, 21, 55, 64
Vishnevsky, B 20, 61
Viveiros, Miguel 11, 18, 24, 27, 53, 79, 92
Vladimyrsky, Michail 27, 93
Voit, Antje 22, 27, 67, 90
von Groll, Andrea 20, 59
Vorobyeva, Alena 32, 115
Vyazovaya, Anna 12, 19, 20, 21, 55, 61, 64
Walker, Timothy 8, 14
Walzl, Gerhard 12, 15, 38
Wangh, L. 28, 96
Warren, Rob 9, 16, 20, 25, 28, 44, 59, 83, 96
Washington Reyes, Carlos 25, 82
Werngren, Jim 26, 88
Wilmanns, Matthias 12, 19, 57
Woolliams, John 9, 17, 45
Wright, David 9, 17, 24, 45, 77
Yablonsky, P 20, 61
Yakunova, Olga 12, 19, 58
Yaman, Gorkem 29, 99
Yeboah-Manu, Dorothy 40
Yegenoglu, Yildiz 29, 100
Young, Douglas 40
Zaha, Arnaldo 20, 59
Zajc, Urska 30, 107
Zambrano, Maria Mercedes 9, 16, 41, 42
Zambrano, Magda Lorena Orduz 23, 75
Zdelar-Tuk, Maja 21, 65
Zele, Diana 30, 107
Zhuravlev, Viacheslav 20, 26, 27, 61, 88, 94
Zolnir Dovc, Manca 21, 62
Zoltán, Lőrinczi 27, 93 Zomer, Aldert 22, 68
123

Florence 2013 - European Society of Mycobacteriology

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