European Society of Mycobacteriology

ESM

In co-organization with the Instituto Nacional de Saúde Dr. Ricardo Jorge CONTENTS

• Welcome Message • Introduction to the European Society of Mycobacteriology • Congress Organization • Program at a glance • Programme of Guest Lectures, Oral Presentations and Symposia • Programme of Poster Presentations • Abstracts of Guest Lectures (GL) • Abstracts of Oral Presentations (OP) • Abstracts of Poster Presentations (PP) • Author Index

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 1 Welcome Message

Dear Colleagues,

It is with great pleasure that we warmly welcome you to the 30th Annual Congress of the

European Society of Mycobacteriology, held in the city of Porto in Portugal from July 5-8, 2009.

The aim of ESM2009 is to promote the exchange of distinguished experts from all around

the world who will have the opportunity to update information in the front-line of scientific

achievement, share experience and ideas, and actively participate in contribution to the field of

mycobacteriology.

We have worked hard to make your stay in Porto a pleasant, useful and memorable occasion.

On behalf of the ESM and the Local Organisers,

Suzana David

2 ESM 2009 European Society of Mycobacteriology 30th Annual Congress July 5-8, 2009 Porto, Portugal

The European Society of Mycobacteriology (ESM, http://www.esmycobacteriology.eu), founded in 1980, is a non-profit international scientific society dealing with different aspects of mycobacteriology and related diseases. It is considered one of the most active international scientific societies in this area, being committed to: - Encouraging the highest standards for research to facilitate the discovery of new knowledge; - Coordinating and providing information and expertise to other organizations worldwide; - Disseminating knowledge on all aspects of mycobacteriology and related diseases, through scientific meetings and publications, - Encouraging and providing the highest standards of training to interested health care providers; - Establishing, reviewing and revising guidelines; - Promoting high quality and cost effective diagnostic procedures; - Advising, cooperating and participating with government and non-government agencies in matters of common interest; - Participating in activities whose aim is to prevent mycobacterial diseases worldwide.

The ESM meetings are held each year in a different country of Europe. The ESM2009 congress is hosted in the city of Porto, in Portugal. International specialists will treat themes at the front-line of scientific achievement in the field of mycobacteriology and related diseases. As in previous meetings, the scientific program of the conferences covers a wide range of topics in both applied and fundamental research in areas of priority such as: - Diagnostics of active and latent tuberculosis - Diagnostics of atypical mycobacteria - Molecular epidemiology of tuberculosis - Antibiotic resistance, MDR, XDR - Tuberculosis drug development - Immunology of mycobacterial infections - Molecular Biology of mycobacteria - Taxonomy of the genus Mycobacterium - Veterinarian and environmental Mycobacteriology - Methods for diagnostics adapted to economically disfavoured settings - Laboratory safety - Short and long term programs and recommendations

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 3 Congress Organization

EUROPEAN SOCIETY OF MYCOBACTERIOLOGY http://www.esmycobacteriology.eu Chairman of the ESM 2009 congress Suzana David (Lisbon, Portugal)

Steering Committee General Secretary Enrico Tortoli (Firenze, Italy) President Todor Kantardjiev (Sofia, Bulgaria) Treasurer Malcolm Yates (East Dulwich Grove, U.K.) Members Maria-Jesus Garcia (Madrid, Spain) Sven Hoffner (Solna, Sweden) Stefan Niemann (Borstel, Germany) Gabriela Pfyffer (Luzern, Switzerland) Nalin Rastogi (Guadeloupe, France) Veronique Vincent (Geneva, Switzerland)

HONORS COMMITTEE ESM 2009

Ana Jorge Minister of Health Mariano Gago Minister of Science, Technology and Higher Education Manuel Pizarro Secretary of State for Health Francisco Ramos Secretary of State for Health José Pereira Miguel President of the National Health Institute Dr. Ricardo Jorge Jorge Sampaio UN Secretary General’s Special Envoy to Stop TB Mario Raviglione Director of the Stop TB Department, World Health Organization Maria do Céu Machado High Comissioner for Health Francisco George Director-General of Health Jorge Soares Director of the Health and Human development Service Calouste Gulbenkian Foundation Jorge Torgal Director do Institute of Hygiene and Tropical Medicine Rui Rio Mayor of the city of Porto Fernando Augusto Fiuza de Melo Director of the Clemente Ferreira Institute, São Paulo, Brasil

4 ESM 2009 COORGANIZERS

European Society of Mycobacteriology http://www.esmycobacteriology.eu

Instituto Nacional de Saúde Dr. Ricardo Jorge, INSA http://www.insa.pt

PARTNERS

Foundation for Science and Technology (FCT) http://www.fct.mctes.pt

Fundação Calouste Gulbenkian http://www.gulbenkian.pt

Luso-American Foundation (FLAD) http://www.flad.pt

STOP-TB Working Group on New Diagnostics, Point of Care sub group http://www.stoptb.org

SPONSORS The Organization expresses its thanks and appreciation to all those who gener- ously contributed to the success of the 30th Annual Congress of ESM.

HAIN LifeSciences http://www.hain-lifescience.com Platinum Medal Sponsor

Beckton Dickinson & Quilaban http://www.bd.com http://www.quilaban.pt Gold Medal Sponsor

BioMerieux http://www.biomerieux.com Gold Medal Sponsor

Microsens (logo) http://www.microsens.com

Cepheid http://www.cepheid.com

Genoscreen http://www.genoscreen.com

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 5 Program at a glance

Sunday, July 5th 09h00 REGISTRATION 11h00 – 12h00 SYMPOSIUM SPONSORED BY HAIN LIFESCIENCE GMBH: RAPID MOLECULAR GENETIC DETECTION OF MDR- AND XDR-TB Chair: Michael Weizenegger Speakers: Doris Hillemann and Christopher M Gilpin 12h00 – 13h30 SYMPOSIUM SPONSORED BY STOP-TB WORKING GROUP ON NEW DIAGNOSTICS, POINT OF CARE SUB GROUP: A SYMPOSIUM ON POINT-OF-CARE TESTS FOR TUBERCULOSIS Speakers: Catharina Boehme, Carol Nawina Nyirenda, Rosanna Peeling, Gerd Michel, Ruth McNerney and Amy P Wong 13h30 – 15h30 Break for Lunch 15h30 – 16h30 SYMPOSIUM CO-SPONSORED BY BECTON, DICKINSON AND COMPANY AND QUILABAN - QUÍMICA LABORATORIAL ANALÍTICA LDA: SUCCEPTIBILITYTESTING AND TREATMENT OF TB IN THE ERA OF MDR-TB AND XDR-TB. DO WE USE THE RIGHT DRUGS AND TOOLS? Chair: Francoise Portaels and Salman Siddiqi Speakers: Stefan Winkler, Andre De Bock, and Virginia Crews 17h00 – 19h00 OPENING SESSION Welcome Address

TB CONTROL PROGRAMS Chair: Jaime Nina and Cristina Furtado Guest Lecture-1: Miguel Villar Guest Lecture-2: Fernando Fiuza de Melo 19h15 Welcome Reception

Monday, July 6th 09h00 – 13h15 SCIENTIFIC SESSION ON MOLECULAR EPIDEMIOLOGY AND DRUG RESISTANCE SURVEILLANCE Chair: Stefan Niemann and Cristina Gutierez 9h00 – 9h45 Guest Lecture-3: Sebastien Gagneux 9h45 – 11h00 Oral Presentations 11h00 – 11h30 Coffee and tea Chair: Nalin Rastogi and Dr. Philip Supply 11h30 – 12h15 Guest Lecture-4: Dick van Soolingen 12h15 – 13h015 Oral Presentations 13h15 – 14h00 Lunch

6 ESM 2009 14h00 – 15h00 POSTER SESSION 15h00 – 16h45 SCIENTIFIC SESSION ON NON TUBERCULOUS MYCOBACTERIA Chair: Enrico Tortoli 15h00 – 15h45 Guest Lecture-5: Joseph O. Falkinham, III 15h45 – 16h45 Oral Presentations 16h45 – 17h15 Coffee and tea 17h15 – 19h15 SCIENTIFIC SESSION ON ISSUES IN THE MODERN TB LABORATORY Chair: Gabriela Pfyffer and Thomas Shinnick 17h15 – 18h00 Guest Lecture-6 : Dr. Jean-Pierre Zellweger 18h00 – 18h30 Guest Lecture-7 : Lee W. Riley 18h30 – 19h15 Oral Presentations 19h30 Visit and Dinner at the Port Wine Cellars

Tuesday, July 7th 09h00 – 12h30 SCIENTIFIC SESSION ON VACCIN DEVELOPMENT AND PATHOGENESIS Chair: David Minnikin 09h00 – 09h45 Guest Lecture-8: Carlos Martin 09h45 – 10h30 Guest Lecture-9 : Lee W. Riley 10h30 – 11h00 Coffee and tea 11h00 – 12h30 Oral Presentations 12h30 – 13h15 Lunch 13h15 – 14h15 BUSINESS MEETING 14h15 – 18h15 SCIENTIFIC SESSION ON LABORATORY STRENGTHENING Chair: Dr. Sven Hoffner and Dr. Mark Perkins 14h15 – 15h00 Guest Lecture-10: Thomas Shinnick 15h00 – 15h45 Guest Lecture-11: Afrânio Kritski 15h45 – 16h30 Guest Lecture-12 : Moisés Palaci 16h30 – 17h00 Coffee and tea 17h00 – 17h45 Oral Presentations 17h45 – 18h15 BEST POSTER 18h30 Cultural Evening Dinner

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 7 Wednes- 09h00 – 11h00 SCIENTIFIC SESSION ON DRUG DEVELOPMENT day, July 8th Chair: Dr. Nalin Rastogi 09h00-09h45 Guest Lecture-13: Clarice Queico Leite 09h45 – 10h30 Guest Lecture-14 : Moisés Palaci 10h30 – 11h00 Oral Presentations 11h00 – 11h30 Coffee and tea 11h30 – 12h20 SESSION ON PRACTICAL ASPECTS AND QUALITY ASSURANCE IN MOLECULAR EPIDEMIOLOGY Chair: Dr. Elvira Richter and Prof. Christophe Sola 11h30 – 12h00 Guest Lecture-15 : Philip Supply 12h00 – 12h20 Oral Presentation 12h20 – 12h30 Discussion 12h30 – 13h30 SYMPOSIUM SPONSORED BY INSTAND, SOCIETY FOR RESEARCH PROMOTION OF QUALITY ASSURANCE IN MEDICAL LABORATORIES, WHO COLLABORATING CENTRE, DUESSELDORF, GERMANY: EXTERNAL QUALITY ASSURANCE Speakers: Elvira Richter, Girts Skenders, Akos Somoskövy 13h:30 – 13h45 Closing Remarks 13h45 – 14h15 CLOSING OF CONGRESS 14h15 Conference Closes

8 ESM 2009 Programme of Guest Lectures, Oral Presentations and Symposia

Sunday, July 5th

9h00 – 11h00 REGISTRATION 11h00 – 12h00 SYMPOSIUM SPONSORED BY HAIN LIFESCIENCE GMBH RAPID MOLECULAR GENETIC DETECTION OF MDR- AND XDR-TB Chair: Dr. Michael Weizenegger Dr. Doris Hillemann, National Reference Center for Mycobacteria (Borstel, Germany) Rapid detection of XDR-TB with the Genotype MTBDRsl assay Dr. Christopher M Gilpin, PhD MPH, International Organization for Migration (Geneva, Switzerland) Implementation of new diagnostic tools and algorithms for enhanced case detection of drug resistant forms of tuberculosis 12h00-13h30 SYMPOSIUM SPONSORED BY STOP-TB WORKING GROUP ON NEW DIAGNOSTICS, POINT OF CARE SUB GROUP A SYMPOSIUM ON POINT-OF-CARE TESTS FOR TUBERCULOSIS Dr. Catharina Boehme, M.D., Foundation for Innovative Diagnostics (FIND) (Geneva, Switzerland) What is a POC test? Carol Nawina Nyirenda (Zambia) Why do we need rapid tests: a patient’s perspective? Prof. Rosanna Peeling, Ph.D., London School of Hygiene & Tropical Medicine (London, UK) Rapid tests for TB: what is wrong with them? Dr. Gerd Michel, Ph.D., Foundation for Innovative Diagnostics (FIND) (Geneva, Switzerland) Biomarker discovery: are we making progress Dr. Ruth McNerney, Ph.D., London School of Hygiene & Tropical Medicine (London, UK) Volatile markers for TB: myth or reality? Dr. Amy P Wong, Ph.D., X PRIZE Foundation (California, U.S.A.) Barriers to TB test development Discussion: The way forward Platform and floor 13h30 – 15h30 Break for Lunch 15h30 – 16h30 SYMPOSIUM CO-SPONSORED BY BECTON, DICKINSON AND COMPANY AND QUILABAN - QUÍMICA LABORATORIAL ANALÍTICA LDA SUCCEPTIBILITYTESTING AND TREATMENT OF TB IN THE ERA OF MDR-TB AND XDR-TB. DO WE USE THE RIGHT DRUGS AND TOOLS? Chair: Prof. Francoise Portaels and Dr. Salman Siddiqi Prof. Stefan Winkler, University of Vienna (Vienna, Austria) Antibiotic therapy for TB and new treatment options European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 9 Dr. Andre De Bock (BD) Extended Drug Susceptibility Testing of M. tuberculosis Virginia Crews (BD) The new BD MGIT TBc Identification Test 16h30 – 17h00 OPENING SESSION WELCOME Minister of Health (Portugal) (to confirm) Minister of Science, Technology and Higher Education (Portugal) (to confirm) 17h00 – 17h45 Welcome from the President of the Instituto Nacional de Saúde Dr. Ricardo Jorge, Prof. José Pereira Miguel (Portugal) Welcome from the General Secretary, Dr. Enrico Tortoli (Italy)

Welcome address, Dr. Suzana David (Portugal)

TB CONTROL PROGRAMS Chair: Prof. Jaime and Prof Cristina Furtado 17h45 – 18h20 Guest lecture: Dr. Miguel Villar, Consultant or Thuberculosis, Directorate-General of Health (Lisboa, Portugal) Tuberculosis in Portugal 18h20 – 19h00 Guest lecture: Dr. Fernando Fiuza de Mello, Director of the Clemente Ferreira Institute (São Paulo, Brasil) The Brazilian experience in the control of multi-drug resistant tuberculosis Welcome reception Welcome Reception

Monday, July 6th SCIENTIFIC SESSION ON MOLECULAR EPIDEMIOLOGY AND DRUG RESISTANCE SURVEILLANCE 9h00 – 9h45 Guest lecture: Dr. Sebastien Gagneux, Ph.D., National Institute for Medical Research GL-3 (London, UK) Evolutionary forces in Mycobacterium tuberculosis Chair: Dr. Stefan Niemann and Dr. Cristina Gutierez 9h45 – 10h00 Honisch C, Mosko M, Arnold C, Gharbia S, Feuerriegel S, Niemann S OP-1 Mass Spectrometry for molecular typing of the Mycobacterium tuberculosis complex: One platform and multiple assay formats 10h00 – 10h15 Borile C, Refrégier G, Labarre M, Franz S, Mézard M, Sola C OP-2 Clustering of spoligo-patterns: towards an automation of Mycobacterium tuberculosis complex classification 10h15 – 10h30 Sandoval A, Cubillos A, Reyes A, Correa N, Robledo J, Zambrano MM, OP-3 Del Portillo P Identification of the insertion element IS6110 in phop promoter in a high transmission Mycobacterium tuberculosis strain: A clue to phenotypic variation 10h30 – 10h45 Oelemann MC, Gomes HM, Willery E, Lima KVB, Possuelo L, Locht C, Goguet de OP-4 la Salmonière YOL, Gutierrez MC, Supply P, Suffys PN

10 ESM 2009 Genomic interrogation of mycobacterium tuberculosis isolates from Brazil 10h45 – 11h00 Mokrousov I, Valcheva V, Sovhozova N, Aldashev A, Rastogi N, Isakova J OP-5 Penitentiary population of Mycobacterium tuberculosis in Kyrgyzstan: Exceptionally high prevalence of the Beijing genotype and its Russia-specific subtype 11h00 – 11h30 Coffee and Tea 11h30 – 12h15 Guest lecture: Dr. Dick van Soolingen, Ph.D., National Institute for Public Health and GL-4 the Environment (Bilthoven, Netherlands) Advances in the molecular epidemiology of tuberculosis Chair: Dr. Nalin Rastogi and Dr. Philip Supply 12h15 – 12h30 Millet J, Miyagi-Shiohira C, Yamane N, Mokrousov I, Rastogi N OP-6 The unique endemic nature of Beijing genotype strains in Okinawa, Ryukyu Islands of Japan as revealed by newly described 15 and 24-loci MIRU-VNTR typing schemes 12h30 – 12h45 Shamputa IC, Lee J, Allix-Béguec C, Cho E-J, Lee J-I, Min JH, Goldfeder LC, Kim JH, OP-7 Kang HS, Hwang SH, Eum SY , Lee H, Park SK, Supply P, Cho SN, Via LE, Barry III CE Mycobacterium tuberculosis genetic diversity in South Korea

12h45 – 13h00 Feuerriegel S, Homolka S, Post E, Oberhauser B, George AG, Westman L, Dafae F, OP-8 Rüsch-Gerdes S, Niemann S Correlation of molecular resistance mechanisms and phenotypic resistance to first-line drugs in Mycobacterium tuberculosis strains from Sierra Leone 13h00 – 13h15 McNerney R, Mallard K OP-9 Lam and HIV: correlation or co-incidence? 13h15 – 14h00 Lunch 14h00 – 15h00 POSTER SESSION SCIENTIFIC SESSION ON NON TUBERCULOUS MYCOBACTERIA Chair: Dr. Enrico Tortoli 15h00 – 15h45 Guest lecture: Prof. Joseph O. Falkinham, III, Ph.D., Virginia Tech GL-5 (Virginia, U.S.A.) Surrounded by mycobacteria 15h45 – 16h00 Lyberopoulos P, Frangopoulos F, Kontos F, Zerva L, Malagari Ai, Papiris S OP-10 Mycobacterium celatum: An emerging pathogen in the immunocompetent. A case report

16h00 – 16h15 Radomski N, Thibault V, Karoui C, De Cruz K, Cochard T, Gutiérrez C, Supply P, OP-11 Biet F, Boschiroli ML Mycobacterium avium subspecies strains from human and animal origin 16h15 – 16h30 Leão SC, Tortoli E, Viana-Niero C, Ueki SYM, Lima KVB, Lopes ML, Yubero J, OP-12 Menendez MC, Garcia MJ The characterization of mycobacteria from an outbreak suggests a revision of the taxonomic status of members of the Mycobacterium chelonae-abscessus group

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 11 16h30 – 16h45 Santos R, Marques M, Oliveira P, Carvalho F, Carvalho C, Monteiro G, Cabral J, OP-13 Frade R, Silva M, Fernandes P The sunny side of mycobacteria 16h45 – 17h15 Coffee and Tea SCIENTIFIC SESSION ON ISSUES IN THE MODERN TB LABORATORY Chair: Dr. Gabriela Pfyffer and Dr. Thomas Shinnick Guest lecture: Dr. Jean-Pierre Zellweger, M.D., Swiss Lung Association (Bern, Switzerland) 17h15 – 18h00 The use of Interferon Gamma Release Assays as an aid in the con- GL-6 trol of tuberculosis 18h00 – 18h30 Guest lecture: Prof. Lee W. Riley, M.D., Ph.D., University of California at Berkeley GL-7 (California, U.S.A.) A novel diagnostic test to differentiate latent TB infection and active disease

18h30 – 18h45 Kaal E, Kolk A, Kuijper S, Janssen HG OP-14 Fast identification of Mycobacterium tuberculosis in sputum and cultures based on thermally-assisted hydrolysis and methylation by gas chromatography-mass spectrometry 18h45 – 19h00 Ängeby K, Juréen P, Giske C, Chryssanthou E, Werngren J, Hoffner S, Kahlmeter OP-15 G, Sturegård E, Schön T How Wild-type MIC distributions can be useful to determine clinical breakpoints in Mycobacterium tuberculosis 19h00 –19h15 Miotto P, Cirillo DM OP-16 Molecular techniques to monitor TB patients’ treatment: selective removal of DNA from dead bacteria in mixed populations by use of ethidum monoazide Visit and Dinner at the Port Wine Cellars

Tuesday, July 7th SCIENTIFIC SESSION ON VACCIN DEVELOPMENT AND PATHOGENESIS Chair: Dr. David Minnikin 9h00 – 9h45 Guest lecture: Prof. Carlos Martin, M.D., Ph.D., University of Zaragoza GL-8 (Zaragoza, Spain) New live tuberculosis vaccines strategies 09h45 – 10h30 Guest lecture: Prof. Lee W. Riley, M.D., Ph.D., University of California at Berkeley GL-9 (California, U.S.A.) Regulation of Mycobacterium tuberculosis cell wall lipid composition and its effect on in vivo bacterial persistence 10h30 – 11h00 Coffee and Tea 11h00 – 11h15 Brzostek A, Pawelczyk J, Rumijowska-Galewicz A, Dziadek B, Dziadek J OP-17 Mycobacterium tuberculosis is able to accumulate and utilize cholesterol 11h15 – 11h30 Rodríguez-Güell E, Alonso C, del Val-Romero B, Clivillé R, Secanella SP, Roura-Mir OP-18 C, Cañete C, Navarro A, de Gispert FX , Luquin M, Julián E Mycolic acid-induced IFN-g production by CD1-restricted T cells from tuberculous patients 11h30 – 11h45 Farnia P, Ali Veleyati A, Masjedi MR, Ibrahim TA, Tabarsei P, Haroun RZ, Kuan HO, OP-19 Omar AR A report on new adapted forms of extensively drug resistance tubercle bacilli : Transmission Electron Microscopy analysis 12 ESM 2009 11h45 – 12h00 Homolka S, Niemann S, Russell DG, Rohde KH OP-20 Growth profile of clinical isolates of Mycobacterium tuberculosis complex in murine macropghages 12h00 – 12h15 van Ingen J, van der Wel N, Dekhuijzen R, Boeree M, van Soolingen D OP-21 Presence of esat-6 and cfp-10 genes does not lead to phagolysosome translocation of Mycobacterium szulgai 12h00 – 12h30 Fraga AG, Braga JE, Cruz A, Martins TG, Pereira DR, Meyers WM, Portaels F, OP-22 Castro AG, Pedrosa J Development of an adaptive immune response in the draining lymph node during Mycobacterium ulcerans infection 12h30 – 13h15 Lunch 13h15 – 14h15 BUSINESS MEETING SCIENTIFIC SESSION ON LABORATORY STRENGTHENING Chair: Dr. Sven Hoffner and Dr. Mark Perkins

14h15 – 15h00 Guest lecture: Dr. Thomas M. Shinnick, Ph.D., Centers for GL-10 Disease Control and Prevention (Georgia, U.S.A.) CDC’s global TB laboratory activities 15h00 – 15h45 Guest lecture: Prof. Afranio Kritski, M.D., Ph.D., Universidade Federal do Rio de GL-11 Janeiro (Rio de Janeiro, Brazil) Development and validation of new TB diagnostic tests in Brazil: experience of Rede-TB 15h45 – 16h30 Guest lecture: Prof. Moisés Palaci, Ph.D., Universidade Federal do Espírito Santo GL-12 (Vitória, Brazil) Experience of a successful mycobacteriology laboratory network in Espirito Santo- Brazil 16h30 – 17h00 Coffee and Tea 17h00 – 17h15 Portugal C, Cardoso N, Sancho L, Sousa G OP-23 Tuberculosis software in a general hospital, working instrument 17h15 – 17h30 den Hertog A, Koeleman M, Ingham C, Fey F, Langerak E, Klatser P, Anthony R OP-24 Development of an automated culture system for M. tuberculosis with autofluorescence detection 17h30 – 17h45 Morcillo N, Imperiale B, Di Giulio B OP-25 Second-line drug susceptibility testing of Mycobacterium tuberculosis by MGIT 960 system, the microplate colorimetric-based method and the proportion method 17h45 – 18h15 BEST POSTER Cultural Evening Dinner

Wednesday, July 8th SCIENTIFIC SESSION ON DRUG DEVELOPMENT Chair:Dr. Nalin Rastogi 9h00 – 9h45 Guest lecture: Prof. Clarice Queico Fujimaro Leite, M.Sc., Ph.D.; Universidade GL-13 Estadual Paulista (Araraquara, Brazil)

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 13 Screening of molecules with anti-TB activity, from the Brazilian cerrado plants, and synthetic metallo- organic compounds 09h45 – 10h30 Guest lecture: Prof. Moisés Palaci, Ph.D., Universidade Federal do Espírito Santo GL-14 (Vitória, Brazil) Clinical trials of drugs and diagnostic tests: the challenges in mycobacteriology 10h30 – 10h45 van Ingen J, Boeree M, Amaral L, Pando RH, van Soolingen D OP-26 Thioridazine shows promising activity in a murine model of multidrug-resistant tuberculosis 10h45 – 11h00 Rodrigues L, Sampaio D, Couto I, Machado D, Kern WV, Amaral L, Viveiros M OP-27 Contribution of efflux pump activity for macrolide resistance in M. avium complex 11h00 – 11h30 Coffee and Tea SESSION ON PRACTICAL ASPECTS AND QUALITY ASSURANCE IN MOLECULAR EPIDEMIOLOGY Chair: Dr. Elvira Richter and Prof. Christophe Sola 11h30 – 12h00 Allix-Béguec C, HubansC, Ferreira S, Supply P GL-15 New, easy-to-use tools for quality-controlled genotyping of M. tuberculosis complex strains 12h00 – 12h20 Abadia E, Zhang J, Refrégier G, Sola C OP-28 Membrane- based versus microbead- based spoligotyping: Preliminary results on a quality- insurance study on 10 sites worlwide 12h20 – 12h30 Discussion SYMPOSIUM SPONSORED BY INSTAND, SOCIETY FOR RESEARCH PROMOTION OF QUALITY ASSURANCE IN MEDICAL LABORATORIES, WHO COLLABORATING CENTRE, DUESSELDORF, GERMANY 12h30 – 13h30 EXTERNAL QUALITY ASSURANCE PD Dr. Elvira Richter, NRL (Borstel, Germany) EQA in a low incidence, high income country Dr. Girts Skenders, State Agency of TB and Lung Disease (Latvia) Organization and EQA of Latvian TB laboratory network Dr. Akos Somoskövy, M.D., Ph.D., D.Sc., Foundation for Innovative Diagnostics (FIND) (Geneva, Switzerland) Quality Assurance for new techniques – what is necessary, what is possible? 13h30 -13h45 Closing remarks 13h45 – 14h15 CLOSING OF CONGRESS

14h15 Conference closes

14 ESM 2009 Programme of Poster Presentations

Miotto P, Baldan R, Cirillo DM PP-1 Evaluation of the high-throughput repetitive-sequence-based PCR diversilab system in M. tuberculosis molecular epidemiology studies Zhang J, Abadia E, Refrégier G, Ruimy R, Boschiroli ML, Guillard B, Sola C PP-2 68 spacers Mycobacterium tuberculosis complex spoligotyping : A study using a microbead-based high throughput format Niemann S, Khechinashvili G, Gegia M, Mdivani N, Tang YW PP-3 Association between Beijing genotype and drug resistance among Mycobacterium tuberculosis isolates circulating in the Republic of Georgia Baboolal S, Millet J, Akpaka PE, Ramoutar D, Rastogi N PP-4 Mycobacterium tuberculosis epidemiology and genetic diversity in the Twin Island Republic of Trinidad and Tobago Mestre O, Luo T, Rauzier J, Golec M, Rastogi N, Rasolofo V, Tonjum T, Sola C, Matic I, Mei J, Gao Q, Vultos PP-5 TD, Gicquel B Diversity and evolution of M. tuberculosis Sharaf-Eldin GS, Elmoula IF, Ali MS, Saaed NS, Ali AB, Mallard K, McNerney R, Algamdi S PP-6 Spoligotype patterns and drug resistant profile of Mycobacterium tuberculosis in Sudan Valcheva V, Mokrousov I, Panaiotov S, Bachiiska E, Zozio T, Sola C, Markova N, Rastogi N PP-7 Controversial dissemination pattern of the Bulgaria-specific M. tuberculosis spoligotype ST125_BGR Panaiotov S, Bachiyska E, Brankova N, Levterova V PP-8 Mycobacterium tuberculosis Beijing genotype and origins of the Bulgarians Al-Maniri AA, Singh JPN, Al-Rawas O, Al Busaidi S, Al Balushi L, Ahmed I, Al- Mahruqi S, Haile PP-9 M, Diwan V, Hoffner S A Snapshot on biodiversity and clustering of Mycobacterium tuberculosis among nationals and immigrants in Oman using spoligotyping David S, Ribeiro JN, Maio JN, João I, Amorim A, Pereira E PP-10 The extent of the Latin American-Mediterranean Mycobacterium tuberculosis spoligotype family in Portugal Von Groll A, Martin A, Felix C, Prata P, Honscha G, Portaels F, Almeida da Silva P, Palomino JC PP-11 Fitness Study of the RDRio lineage and LAM family of Mycobacterium tuberculosis in a study population in Rio Grande, Brazil Perdigão J, Silva C, Portugal I PP-12 Genomic characterization of Lisboa family strains by deletion analysis Obrovac M, Katalinic-Jankovic V, Grce M, Zmak L PP-13 Importance of molecular typing in suspected intra-familial transmission of tuberculosis Oral Zeytinli U, Kayar Mb, Karacali A, Sahan Kipalev A, Yula E, Köksal F PP-14 Detection of clonal complexity in clinical M. tuberculosis Isolates by MIRU-VNTR in Cukurova Region, Turkey Leite CQF, Santos ACB, Pandolfi JRC, Malaspina AC, Pavan FR, Mendes NH, Viana BHJ PP-15 Molecular epidemiology study of tuberculosis patients in a small city of São Paulo – Brazil, from 2002 to 2006 Leite CQF, Nogutia EN, Malaspina AC, Santos ACB, Hirata RDC, Hirata MH, Cardoso RF PP-16 Genotyping of Mycobacterium tuberculosis in northwest of Paraná State of Brazil

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 15 Mello FAF, Albarral MIP, Mendes NH, Pandolfi JRC, Santos ACB, Almeida EA, Cardoso RF PP-17 Spoligotyping of Mycobacterium tuberculosis isolated from patients of Clemente Ferreira ambulatory in São Paulo, SP – Brazil Tajeddin E, Farnia P, Kargar M, Noroozi J, Ahmadi M, Kazempour M, Hadadi M, Masjedi M, Velayati A PP-18 Comparison Of Mycobacterium Beijing Genotype with VNTR, Spoligotyping and RFLP-IS6110 Ritacco V, Reniero A, Beltrán M, López B, Kantor I, Barrera L PP-19 Multiply recurrent tuberculosis in a pacient living with HIV: Reinfection or reactivation? Tavares Magalhães A, Alves A, BragaR, Valente I, DuarteR, Miranda A PP-20 Molecular epidemiology of tuberculosis in Vila Nova de Gaia, Portugal Alves A, Miranda A PP-21 Molecular study of recurrent tuberculosis cases Ehricht R, Slickers P, Monecke S PP-22 Genotyping of drug resistance in Mycobacterium tuberculosis using diagnostic microarrays Al-Hajoj S, Varghese B, Herbawi M, Al-Omari R, Allix-Béguec C PP-23 Genotyping of mono and multi-drug resistance TB in Saudi Arabia Machado D, Viveiros M, Rodrigues L, Couto I , Amaral L PP-24 Early detection of MDRTB by molecular tools in the control of drug resistant tuberculosis in Portugal: a case of success Vladimirov K, Zaitseva E, Ivanov A PP-25 Drug-resistance of Mycobacterium tuberculosis at penitentiary institutions of St. Petersburg, Russian Federation Stoffels K, Fauville-Dufaux M PP-26 An increase of drug resistance since 2001 in multidrugresistant M. tuberculosis isolates from Belgium Perdigão J, Ferreira A, Malaquias A, Macedo R, Brum L, Portugal I PP-27 Mutational analysis of genes associated with resistance to injectable second-line drugs in Mycobacterium tuberculosis clinical isolates from Lisbon, Portugal Nuak J, Ferreira D, Carvalho T, Gomes MH, Sarmento A PP-28 Multidrug-resistant tuberculosis Tudó G, Rey E, Alcaide F, Coll P, Codina G, Martín-Casabona N, Montemayor M, Moure R, Salvadó M, PP-29 González-Martín J Characterisation of streptomycin mutations in Mycobacterium tuberculosis clinical isolates in the area of Barcelona Fattorini L, Pardini M, Cirillo D, Borroni E, Miotto P, Filippini P, Cassone A PP-30 Surveillance of Drug-Resistant Tuberculosis in Italy Sancho L; Portugal C; Tancredo L; Silva M; Dias A; Silva F, Sousa G PP-31 Tuberculosis resistance in a general hospital in Portugal – 9 years surveillance Yates M, Brown T, Drobniewski F PP-32 Does a mutation in the rpoB mean that the M. tuberculosis is resistant to rifampicin? Zaldumbide MA, Mazarrasa CF, Martinez-Martinez L, Balbin JA PP-33 Genotypic detection of isoniazid and rifampin resistance in Mycobacterium tuberculosis clinical isolates Chan CYR, Chan WCE, Au TKM, Lai WMR, Yew WW, Yip CW, Kam KM PP-34 Physiological fitness and transmission potential of multi-drug resistant Mycobacterium tuberculosis clinical isolates in Hong Kong

16 ESM 2009 Sousa AS, Pinheiro MD, Carvalho T, Gonçalves H PP-35 Mycobacterium tuberculosis: 1999-2008 antituberculosis drugs surveillance in clinical isolates from patients in the largest hospital in the North of Portugal Von Groll A, Martin A, Jureen P, Hoffner S, Portaels F, Palomino JC, Almeida da Silva P PP-36 Fitness cost of Mycobacterium tuberculosis clinical isolates resistant to fluoroquinolones Von Groll A, Martin A, Jureen P, Hoffner S, Portaels F, Almeida DA Silva P, Palomino JC PP-37 In vitro activity of ofloxacin, moxifloxacin and gatifloxacin against Mycobacterium tuberculosis by the resazurin colorimetric method Paasch F, Martin A, Docx S, Fissette K, Portaels F, Palomino JC PP-38 Rapid detection of extensively drug-resistant Mycobacterium tuberculosis by the resazurin microtiter assay plate Montoro E, Yzquierdo S, Lemus D, Echemendia M, Takiff H PP-39 Detection of embB gene codon 306 mutations in ethambutol susceptible and resistant Mycobacterium tuberculosis strains Yew WW, Yan SW, Fung SL, Chau CH, Chan Chiu Y PP-40 Tolerance of moxifloxacin in routine clinical treatment of tuberculosis Perdigão J, Sabino A, Milho C, Macedo R, Brum L, Portugal I PP-41 Characterization of gidB gene in Mycobacterium tuberculosis isolates in Lisbon Health Region: role in streptomycin resistance and epidemiological markers Gaile I, Skenders G, Leimane V, Jansone I, Bauskenieks M, Pole I, Baumanis V PP-42 Fluorquinolone resistant Mycobacterium tuberculosis isolates and their molecular characteristics Samper S, Millan I, Lopez-Calleja AI, Gavin P, Lezcano MA PP-43 Design of a rapid method of identification of a highly transmitted strain based on the localization of IS6110 Gutierrez MC, Brosch R, Marceau M, Tap J, Bourdon E, Brisse Smangenot S, Salvignol G, Barbe V, Médigue PP-44 C, Supply P Driving forces on the evolution of the progenitor of M. tuberculosis Ruiz P, Causse M, Zerolo FJ, Gutierrez J, Casal M PP-45 Resistance, MDR and XDR of M. tuberculosis in Spain in the last years Radomski N, Lucas F, Cambau E, Moulin L, Haenn S, Régis M PP-46 Detection of non tuberculous mycobacteria in surface waters: comparison of culture methods Spicic S, Cvetnic Z, Pate M, Duvnjak S, Zdelar-Tuk M, Racic I PP-47 Typing of Mycobacterium avium subsp. avium from different sources using PvuII– PstI–IS901 restriction fragment length polymorphism (RFLP) in Croatia Spicic S, Duvnjak S, Obrovac M, Zdelar-Tuk M, Katalinic-Jankovic V, Racic I, Cvetnic Z PP-48 Tuberculosis in pets and wild animals living in urban environment Spicic S, Cvetnic Z, Pate M, Katalinic-Jankovic V, Duvnjak S, Ocepek M, Zdelar-Tuk M, Krt B PP-49 IS1245-RFLP based genetic relatedness of the Mycobacterium avium subsp. hominissuis strains isolated from humans, animals and environment in Croatia Lucas F, Radomski N, Cambau E, Moulin L, Haenn S, Moilleron R PP-50 Development of real-time PCR assay for quantification of mycobacteria in surface waters Amorim A, Macedo R, Pereira E PP-51 Nontuberculous mycobacteria, isolated from patients with lung disease, from Lisboa e Vale do Tejo region, during 2008

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 17 Lima KVB, Lopes ML, Furlaneto IP, Lima EJC, Conceição EC, de Sousa MS, Costa ARF PP-52 Nontuberculous mycobacteria infections in the State of Pará, Amazon Region, Brazil Jahromi NS, Seif S, Farnia P, Kazempour M, Kargar M, Nowroozi J, Kazempour M, Masjedi M, Velayati A PP-53 Evaluation of Hsp65, Tb ,Sp Regions in Identifying Mycobacterium Other Than Tuberculosis (MOTT); using PCR-RFLP Svensson E, Ridell M, Åkerström M, Andersson E PP-54 Mycobacterium avium alveolitis after cleansing hotel spa whirlpools Couto I, Machado D, Viveiros M, Rodrigues L, Amaral L PP-55 Identification of nontuberculous mycobacteria in clinical samples using molecular methods: A three-year study

Pate M, Ferme D, Žolnir Dovc ^ M, Ocepek M PP-56 Mycobacteria in animals in Slovenia – an overview of the last decade Leite SRA, Silva P, Sato DN, Santos ACB, Miyata M, Leite CQF PP-57 Isolation and identification of Rhodococcus and Nocardia genders in sputum samples with tuberculosis suspect Neonakis IK, Kontos F, Gitti Z, Baritaki S, Bazigos S, Mihailelis E, Zerva L, Spandidos DA PP-58 PCR-RFLP of hsp65 for identification ofMycobacterium leprae directly from a clinical sample Neonakis IK, Kontos F, Gitti Z, Baritaki S, Kosmadakis G, Baritaki M, Zerva L, Spandidos DA PP-59 A case-report of Mycobacterium thermoresistibile from Greece Portugal C, Sancho L, Dias A, Tancredo L, Silva M; Sardinha T, Sousa JG PP-60 Isolation and frequency of Mycobacterium sp in a general hospital during a 9-year period Diogo J, Rodrigues A, Nascimento I, Sardinha E, Raposo A, Figueira R, Monge I, Silva K, Gil MJ, PP-61 Rodrigues S Laboratory Microbiology contribution to Mycobacterium spp. Diagnosis in three district councils of Setubal (Portugal), an area with high mycobacterial infection prevalence Santos C, Mendes AC, Fernandes SJ, Ramos MH PP-62 Mycobacterium lentiflavum as a causative agent of adenopathy Watson C, Lockwood D PP-63 Teaching old bones new tricks; single nucleotide polymorphism analysis of european archaeological M. leprae DNA Greib C, Lazaro E, Viallard JF, Pellegrin JL, Maugein J PP-64 Interpretation of positive M. tuberculosis antigen specificifnγ release assays in tuberculosis diagnosis Wang S, Neo ZY, Mak KX, Quieng MD, Sing LH PP-65 Direct Identification of Mycobacterium tuberculosis Complex, Mycobacterium avium Complex and Mycobacterium kansasii in Smear-positive Clinical Specimens Müllerova M PP-66 Rapid diagnosis and drug susceptibility testing of tuberculosis infection: MTD- Test2 and Bactec MGIT 960 system Levina K, Dementieva A, Saluotsa M PP-67 First experience with genotype MTBDR assai for rapid evaluation of MDR cases Fajfar N, Zolnir - Dovc M PP-68 Drug resistant tuberculosis in Slovenia and evaluation of genotype MTBDRPLUS test in clinical laboratory

18 ESM 2009 Causse M, Gutierrez-Aroca JB, Casal M PP-69 Evaluation of a new real-time PCR kit for the diagnosis of tuberculosis inrespiratory specimens Karabela S, Papaventsis D, Nikolaou S, Konstantinidou E, Sainti A, Ioannidis P, Kanavaki S PP-70 Quantiferon-TB Gold assay (QFT) and tuberculine skin test (TST) clinical performance for the diagnosis of active tuberculosis Karabela S, Papaventsis D, Nikolaou S, Konstantinidou E, Sainti A, Ioannidis P, Kanavaki S PP-71 Clinical performance of Quantiferon-TB Gold assay (QFT) for the diagnosis of latent tuberculosis in different patient groups Nikolaou S, Karabela S, Papaventsis D, Sainti A, Konstantinidou E, Ioannidis P, Kanavaki S PP-72 Tuberculosis diagnosis by Quantiferon TB Gold assay in areas with differences in TB incidence Havelkova M, Bartu V, Kubin M PP-73 Quantiferon -TB Gold In-Tube test used in Prague patients listed in the National Tuberculosis Register Cacho J, García-Cañas A, González Torralba A, Cano I, Pérez Meixeira A, Ramos Martos, Sánchez- PP-74 Concheiro M Practical experience of using a DNA amplification assay for rapid detection of Mycobacterium tuberculosis complex in respiratory specimens Morgan K PP-75 Real-time polymerase chain reaction for the direct detection of Mycobacterium tuberculosis in clinical specimens Kontos F, Zerva L PP-76 The utility of molecular testing in routine mycobacteriology diagnosis Salas S, Hernández J, Ojeda P, Awad C, de la Hoz F, Murcia M PP-77 Detection of Mycobacterium tuberculosis DNA in formalin-fixed, paraffin-embedded tissue specimens by spoligotyping: application to histopathological diagnosis Cardoso S, Coelho R, Paulo C, Abreu C, Silva S, Gomes H, Sarmento A PP-78 Pott´s Disease: an ancient disease? Loureiro C, Matos G, Balacó I, Mota M, Nogueira C, Lemos S, Rocha G PP-79 Osseous tuberculosis at age of 9 months Secanella SP, Luquin M, Julián E PP-80 Differences in direct antitumoral capacity among the various Mycobacterium bovis BCG substrains Anoosheh S, Farnia P, Noruzi J, Kargar M, Kazempour M, Seif S, Masjedi MR, Velayati AA PP-81 Role of TNF-a gene polymorphisms in host genetic susceptibility to pulmonary tuberculosis Torrado E, Fraga AG, Logarinho E, Martins TG, Carmona JA, Gama JB, Carvalho MA, Proença F, Castro PP-82 AG, Pedrosa J Mycolactone interferes with the protective IFN-γ-dependent activation of macrophages during infection with Mycobacterium ulcerans Montoro E, Valdés I, Aguilar D, Orozco H, Hernández-Pando R PP-83 Virulence, immunogenicity and protection induced by ´Mycobacterium habana´ strains in a murine model of pulmonary tuberculosis Saraiva M, Sousa C, Carmona JA, Cruz A, Pedrosa J, Castro AG PP-84 Dendritic cells differentially express IL12-family cytokines after infection with Mycobacterium tuberculosis or M. bovis BCG Simões MF, Jordão L, Teles JMM, Couto S, Moniz-Pereira J, Pimentel M PP-85 Analysis of M. smegmatis mutants resistant to Ms6 infection

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 19 Julián EG, Rodríguez-Güell E, del Val-Romero B, Clivillé R, Cañete C, Navarro A, de Gispert FX, PP-86 Luquin M, Alonso C Humoral response in tuberculous patients against the mycolic acids of Mycobacterium tuberculosis López AG PP-87 Structural, functional and bioinformatic characterization of TlyA protein from Mycobacterium tuberculosis Ferreira C, Afonso A, Duarte R, Lyashchenko K, Silva A, Rodrigues F, Miranda A, Tavares M, Caldas C, PP-88 Valente F, Valente A, Vasconcelos O, Amado J, Correia-Neves M Evaluation of the applicability of serodiagnosis for tuberculosis in Portugal Martin A, Munga Waweru P, Babu Okatch F, Amondi Ouma N, Bonte L, Palomino JC, Varaine F, Portaels F PP-89 Implementation of the thin layer agar (TLA) for the diagnosis of smear negative pulmonary tuberculosis in a high HIV prevalence setting Ferro RS, Shikama M-L, Villela G, Sato DN, Giampaglia CS, Martins MC, Martin A, Palomino JC PP-90 Direct detection of rifampin resistance in Mycobacterium tuberculosis by the nitrate reductase assay applied directly in sputum samples Ferro RS, Shikama M-L, Villela G, Sato DN, Giampaglia CS, Martins MC, Martin A, Palomino JC, Telles MAS PP-91 Direct detection of rifampin resistance in Mycobacterium tuberculosis by the nitrate reductase assay applied directly in sputum samples De Haas P, Zenhorst R, Mwamba P, Muvwimi M, Mwanza W, Mbulo G, Kapata N, Ayles H PP-92 MTBDRPLUS assay is a useful tool to screen for multi-drug resistant tuberculosis in a national survey de Haas P, Moyoyeta M, Samutela M, Mwanza W, Musunsa A, Mbulo G, Muvwimi M, Ayles H PP-93 Contribution of laboratory factors to high MGIT culture contamination rate in Zambia Ahmed A, Qazi F, Khan AJ PP-94 Programmatic Community-based Management of MDR-TB: Experience in Karachi, Pakistan Muchwa C, Akol J, Mumbowa F, Orikiriza P, Morgan K, Eisenach K, Joloba M, Etwom A, Mugyenyi P, PP-95 Mugerwa R Evaluation of Capilia (TAUNS) for rapid identification ofMycobacterium tuberculosis complex from cultures Muchwa C, Akol J, Orikiriza P, Morgan K, Mumbowa F, Eisenach K, Etwom A, Joloba M PP-96 Comparison of capilia (TAUNS) and IS6110 PCR for rapid identification of Mycobacterium tuberculosis complex from cultures in Kampala, Uganda Bwanga F, Hoffner S, Haile M, Joloba ML PP-97 Direct testing for multi drug resistant tuberculosis with four assays evaluated at Kampala, Uganda McNerney R, Turner C, Mallard K, O’Sullivan D PP-98 PEA production by mycobacteria and its application in a rapid drug susceptibility test Balmoi F PP-99 Various strategies to decontaminate acid fast bacilli positive liquid cultures from Bactec MGIT 960 Orikiriza P PP-100 Low cost isolation of Mycobacterium tuberculosis (MTB) from blood Kayar B, Oral Zeytinli U, Karacali A, Soyal A, Nagiyev T, Köksal F PP-101 Comparison of Rapid Colorimetric Method, Proportion Method and BACTEC460 TB System for testing susceptibility of M. tuberculosis to rifampine and isoniaside

20 ESM 2009 Crews V, Warns M, Pfeltz R, Beaty PS, Rosales J, Kopher K, Joshi S, Hoosen A, Said H PP-102 Evaluation of the MGIT TBc ID test vs two commercially available rapid immunoassays for M. tuberculosis complex organism detection from liquid and solid culture Montoro E, Milián Y, Lemus D, Echemendía M, Yzquierdo S, Martin A, Van der Stuyft P, Palomino JC PP-103 Nitrate reductase assay applied to direct detection of drug resistance in Mycobacterium tuberculosis Montoro E, Lemus D, Madruga M, Mirabal N, Milián Y, Yzquierdo S, Echemendía M, Martín A, Van der PP-104 Stuyft P, Palomino JC Use of nicotinamide in colorimetric methods for rapid detection of pyrazinamide resistance in Mycobacterium tuberculosis Hepple P, Novoa-Cain J, Cheruiyot C, Richter E, Ritmeijer K PP-105 Implementation of liquid culture for tuberculosis diagnosis in a remote setting: Lessons learned Ichijo T, Izumi Y, Yamaguchi N, Nasu M PP-106 Rapid detection of respiratory active mycobacteria by auramine O-CTC double staining Rey E, Tudó G, González-Martín J PP-107 Synergistic activity of two antituberculous drug combinations against clinical isolates of Mycobacterium tuberculosis resistant to isoniazid Stoffels K, Traore H, Van Hoof R, Fauville-Dufaux M PP-108 Tobramycin-clarithromycin combination on Mycobacterium tuberculosis clinical isolates Au-Yeang CKW, Au TK, Chan EWC, Chan RCY PP-109 Prevalence of Efflux-Mediated Rifampicin Resistance in Mycobacterium tuberculosis Clinical Isolates Leite CQF, Pavan FR, Maia PIS, Deflon VM, Sato DN, Azevedo AA, Poelhsitz GV, Leite SRA, Franzblau SG PP-110 Intra and extracellular activity of ruthenium complexes against Mycobacterium tuberculosis and their cytotoxicity Leite S, Pavan F, Maia P, Deflon V, Batista A, Sato D, Franzblau S, Leite C PP-111 Anti-Mycobacterium tuberculosis activity of thiosemicarbazones, semicarbazones and hydrazones Ramos J, Rodrigues L, Couto I, Amaral L, Viveiros M PP-112 Methods for assessment of ethidium bromide transport across Mycobacterium smegmatis cell wall Martins M, Viveiros M, Couto I, Amaral L PP-113 The human macrophage as a model to select compounds active against MDR/XDR-TB Cynamon M, Mookherjee S, Shoen C PP-114 In vitro activities of jpc 2067 alone and in combination with SMX against nocardia species PP-115 Nina J Nosocomial TB in a laboratory setting

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 21

Abstracts of Guest Lectures (GL)

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 23

GL-1

TUBERCULOSIS IN PORTUGAL Miguel Villar Consultant on Tuberculosis, Directorate-General of Health, Lisbon, Portugal

Tuberculosis is a global problem with an estimated number of 9 million cases per year, 83% of which are in Sub-Saharian Africa and South-East Asia, where we find many of the high burden countries. Concerning multidrug-resistance tuberculosis (MDR-TB), WHO estimates about half a million cases globally, per year, including 50.000 extensively drug-resistant tuberculosis (XDR-TB). In 2007, European Union (EU) had an incidence rate of 17/105, having Portugal one of the highest rates in the EU (27/105). In the last 20 years, the incidence in Portugal has decreased consistently more than 7% per year in the last five years. This reduction is mainly in the age group between 25 and 44 years old, leading to a shift to the right of the median age both in the nationals and in the immigrants. The foreign born cases have represented about 12% of the cases and the prevalence of HIV has been around 14%. Most of the cases are pulmonary forms (74.1%) between 2003-2007, 67.5% of which are SS+ and 75.3% are culture positive. Mixed multidrug-resistance tuberculosis, including XDR-TB, during the same period, represents 1.9% (154 cases) of the TB cases at the start of treatment, varying from 1.3% (22 cases) in 2006 to 2.4% (38 cases) in 2004, with an average of 31 cases per year (1.9%). These proportions are representative as the coverage of drug sensibility tests (DST) is over 80%. We will address the importance of the Micobacteriology Laboratory network, concerning case detection, definition of confirmed cases, early diagnosis of MDR-TB and 1st and 2nd line DST. As an important complement of the DOTS Strategy, the analysis of the outcomes will be discussed, concerning not only the general population but also the different risc groups, having as a goal the 85% cure rate proposed by WHO. We finish our presentation addressing the strategy for MDR/XDR-TB control in Portugal, namely the importance of the reference network for MDR-TB with its national coordination.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 25 GL-2

THE BRAZILIAN EXPERIENCE CONTROLING TB MULTI-DRUG RESISTANCE

Fernando Augusto Fiuza de Melo • Médico, Diretor do Instituto Clemente Ferreira – Coordenadoria de Controle de Doenças da Secretaria de Estado da Saúde de São Paulo – ICF/CCD/SP • Doutorado em Medicina, área de pneumologia, pela Escola Paulista de Medicina da Universidade Federal de São Paulo - EPM/UNIFESP • Membro do Comitê de Assessoria Técnico-Científico do Programa Nacional de Controle da Tuberculose do Ministério da Saúde - PNCT/MS

Correspondence: Rua Santo Estácio, 248 – Cidade Vargas, CEP. 04319-010 - Brasil - São Paulo,SP Tele-fax: 0055 11 3218 8653 - Telemóvel 0055 11 8469 4330 - e-mail: [email protected]

Brazil was the first developing country using the short duration regimen of rifampin (R) and isoniazid (H) combined in one capsule for six months, plus pyrazinamide (Z) during the first two months after reorganizing the Brazilian National Tuberculosis Control Program of (BNTBCP, Programa Nacional de Controle da Tuberculose, PNCT) in 1980. At that moment Brazil adopted the anti-TB regimen named E-1 (2RHZ/4RH) for all forms of TB with no known previous treatment. A similar regimen was adopted for Meningitis TB, named E-2, adding corticoids in the intensive phase with the recommendation to lengthen the continuation phase of treatment to 7 months (2RHZCort/7RH). For those TB cases of relapsing (RC) or re-treatment after defaulting (RA) the anti-TB regimen adopted was E-1R with ethambutol (E) during the intensive phase (2RHZE/4RH). The recommended regimen for failure (F-1) cases was named E-3 and included Z and E, associated with streptomycin (S) and the ethionamida (Et) during at least 12 months (3SZEEt/9EEt)¹ The E-1 was evaluated under pragmatic clinical “non-study” conditions during decades 1980/90 and also during half of the first decade of the new millennium and shown an efficacy of 94.6 and 93.9%, an effectiveness of 77.8 and 77.1%, a default rate of 13.7 and 13.1%13%, a failure rate of 1.5 and 1.7%%, a serious adverse events rate of 3.1 and 3.3% and a mortality rate of 3.9 and 4.8%, respectively. (²,³). The small worsening on mortality rate might be related to high rates of defaulting and the HIV co-infection. The good quality of the National Aids Program Control and the increasing rate of TB treatment under direct supervision in Brazil are possible reasons for Brazil rates of resistance were not too high. On the other hand, results of E-3 were not nice with efficacy and effectiveness varying between 57,5% - 85,2 and 66,7 - 84.7%, respectively (4). 4% of 80,000 cases of TB notified in Brazil were resistant to R+H or were unable to be treated with these drugs for any other reason and were defined as F-1 case. These patients were treated with E-3 (3SZEEt/9EEt) and are considered in Brazil as a case of MDR-TB. Brazil estimates a rate between 0,3 and 0,4% of cases of F-1 not responding to E-3. These cases are defined in Brazil as a Multiresistant TB (MRTB) and there is no well established anti-TB regimen for these patients in the BNTBCP5. In 1995 an anti-TB regimen including amicacin (AM), ofloxacin (OFX), terizidona (TRZ), clofazimine (CFZ) and E was evaluated in some TB Centers in Brazil with reasonable results (6). In 2000 the Monitoring Program for MRTB was created, centralizing the notification of all cases of TBMR in Brazil and creating a work group including health professionals involved with MRTB in order to define and organize a way to control MRTB in Brazil. In 2007, Guidelines for MRTB were published presenting the knowledge about TBMR and establishing rules for diagnosis, treatment, prevention and biosafety; providing orientation on epidemiologic surveillance, building human and material resources, and implementing a specific National Notification System for these patients. The current alternative regimen for MRTB cases is defined by the sensitivity tests and administered under supervision, including: AM (or S if sensible) for 12 months, OFX, TRZ and E for 18 months and Z (if sensible) for 6 months7. Metronidazole (MTZ) replacing Z, especially in cases of intolerance or resistance, is used in some clinics. TBMR rates were evaluated in Brazil between 2000 and 2005, showing the following results: Between 323 and 334 cases had been annually notified from 2000 to 2004, with an increase in 2005 to 383 cases notified among a total of 80.000 TB cases. Increasing cure rates over time (40 to 62%), with some organized units showing better rates (75 to 85%).

26 ESM 2009 Default rates between 5 and 7%; failure rates between 10 e 15%; mortality rates had decreased from 33% to 11% over time. Most of MRTB were post-primary cases (74 to 80%); 6 to 8% were the primary cases, especially contacts and risk groups; 11 to 20% were indeterminate. TBMR rate among HIV co-infected patients was low, between 1.6 e 3%7,8.

Extensively multi-drug resistant (X-MDR) TB cases, presenting resistance to 2 first line drugs and 3 second line drugs, have been observed since 20009, however for more accurate estimates, a national survey is necessary. In recent survey at the Clemente Ferreira Institute, 34 cases resistant to fluoroquinolone were notified, and 16 cases were also resistant to AM and 18 to S. The patients were treated with an alternative regimen indicated for MRTB, with 9 cure cases and 25 failure cases, including 17 deaths. An important finding was the occurrence of 3 primary X-MDR cases10.

References Ministério da Saúde/Fundação Nacional de Saúde/Comitê Técnico-Científico de Assessoramento à Tuberculose/Comitê Assessor para Co-infecção HIV-Tuberculose. Tuberculose: guia de vigilância epidemiológica, Brasília, 2002. Ministério da Saúde/Fundação Nacional de Saúde/Centro de Referência Prof. Hélio Fraga-Rio de Janeiro, Documento Básico da Reunião de Avaliação operacional e epidemiológica do PNCT na década de 80. Bol Pneumol Sanit 1993, Numero Especial. Ministério da Saúde/Secretaria de Vigilância em Saúde/Centro de Referência Prof. Hélio Fraga-Rio de Janeiro. Análise da situação da tuberculose no Brasil nos anos 90 e início da década atual. Bol Pneumol Sanit 2005;13:133-179. Campos HS, Melo FAF. Efetividade do esquema 3 (3sSZEEt/9EEt) no retratamento da tuberculose na rotina das unidades de saúde. Bol Pneum Sanit 2000;8:7-14. Melo FAF, Ide Neto J, Seiscento M, Pinto JA, Afiune JB: Tuberculose Multirresistente. J Pneumol 1993;19:73-82. Dalcolmo MP, Fortes A, Melo FAF, Motta R, Ide Neto J, Cardoso N, Andrade M, Barreto AW, Gerhardt G. Estudo de efetividade de esquemas alternativos para o tratamento da tuberculose multirresistente no Brasil. J Pneumol 1999;25:70-77. Ministério da Saúde/Secretaria de Vigilância em Saúde/Centro de Referência Prof. Hélio Fraga/Projeto MSH. Tuberculose multirresistente: guia de vigilância epidemiológica;2005:89pg Melo FAF, Afiune JB, Ide Neto J, Almeida EA, Spada DTA, Antel ANL, Cruz ML. Aspectos epidemiológicos da tuberculose multirresistente em serviço de referência na cidade de São Paulo Rev da Soc Brasil Med Trop 2003;36:733-40. Emergence of Mycobacterium tuberculosis with extensive resistance to second line drugs worldwide 2000 – 2004. MMWR 2006;55(11) Savioli MTG, Melo FAF, Morrone N e Rodrigues DS. Tuberculosis with extensive resistance to drugs in a TB reference center in Sao Paulo, Brazil. Poster accepted for UICTER 2009;Cancum, Mexico.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 27 GL-3

EVOLUTIONARY FORCES IN Mycobacterium tuberculosis

Sebastien Gagneux Division of Mycobacterial Research, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, NW7 1AA, London, United Kingdom; [email protected], phone:+4420 8816-2399, fax: + 4420 8816-2564

The Mycobacterium tuberculosis complex (MTBC) consists of genetically monomorphic organisms. Studying the genetic population structure and evolution of monomorphic bacteria is hindered by the lack of DNA sequence variation; methods such as multilocus sequence typing (MLST), which have been well established in other bacteria, are not applicable. Because of this limitation, most current genotyping methods for MTBC are based on mobile or repetitive DNA elements (e.g. IS6110 RFLP, spoligotyping, MIRU-VNTR). Mobile and repetitive DNA regions change relatively quickly, which makes them ideal markers for molecular epidemiological analyses. However, because these markers can exhibit convergent evolution leading to homoplasy (similar patterns emerging in unrelated strains), they are less robust to infer phylogenetic relationships. Furthermore, actual DNA sequence data is preferred for population genetic analyses. To get around this problem, we sequenced 89 genes in 108 MTBC strains. We used these DNA sequence data to explore the evolutionary forces that have shaped the genetic diversity in MTBC. Our findings show that MTBC is under greatly reduced selective constraint (i.e. purifying selection is reduced in MTBC), and as a result, much of the genetic diversity in MTBC is likely to have functional consequences. These findings have important implications for the development of new tools to control tuberculosis.

28 ESM 2009 GL-4

ADVANCES IN THE MOLECULAR EPIDEMIOLOGY OF TUBERCULOSIS

Dick van Soolingen1, Jakko van Ingen1, Philip Supply2, Anita Schürch1, Ida Parwati3, Reinout van Crevel4, Nguyen Van Hung5, 1- Frank Cobelens6, and Kristin Kremer1 National Tuberculosis Reference Laboratory, Nat. Inst. for Public Health and the Environment (RIVM), Bilthoven, the Netherlands; [email protected] 2 - National Center for Scientific Research, Institut Pasteur, Lille, France 3 - Dept. of Clin. Path. Hasan Sadikin Hosp., Med. Fac. Padjadjaran Univ.,Bandung, Indonesia 4 - Dept. Int. Med., Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands 5 - Nat. Hosp. of Tuberculosis and Respiratory Diseases. Hanoi, Vietnam 6 - Center for Poverty-related Comm. Dis., Acad. Medical Center, Amsterdam, the Netherlands

Although elimination of tuberculosis in Europe is not yet in sight, the ECDC held a meeting in Stockholm in April 2009 to re-define the indicators of a successful TB control on this continent. Molecular epidemiology was identified as a key component to detect the level of active transmission. In 2009, a new ECDC project has been initiated to re-activate the molecular surveillance of (MDR/XDR) TB in Europe, with strong focus on a high coverage of MDR/XDR cases in Central and Eastern Europe. In the previous project transmission of MDR/XDR-TB in Europe was largely caused by Mycobacterium tuberculosis Beijing genotype strains. In a recent (2009) resistance survey in Vietnam, a significant correlation between resistance and Beijing strains was observed. Moreover, in previous studies in Ho Chi Minh City treatment failures and relapses were more frequently found in patients infected by Beijing strains. However, in a recent, larger study in Indonesia patients infected with Beijing genotype strains also more often had a positive sputum culture after six months treatment (RR:1.95; CI 95%:1.25-3.02), but this was not correlated with differences in drug resistance. Therefore, this suggests that M. tuberculosis Beijing genotype strains have a higher capacity to withstand tuberculosis treatment, even in the absence of drug-resistance. The new European network on molecular epidemiology will implement 24-loci VNTR typing as a standard. Although the utility of VNTR typing has been shown in multiple studies, a broad and nation wide comparison of IS6110 RFLP typing and VNTR typing is still missing. In the Netherlands 4400 M. tuberculosis isolates from the period of 2004-2008 have been subjected to IS6110 RFLP as well as VNTR typing and a concordance of 81% has been observed. Moreover, VNTR typing showed a higher degree of concordance with findings in the conventional contact tracing than RFLP typing. To come to the highest resolution of DNA typing, two isolates from the Harlingen tuberculosis outbreak, that have been isolated with an interval of 12.5 years and which were separated by four person-to-person transmissions were subjected to whole genome sequencing. Four single nucleotide polymorphisms (SNPs) and one tandem repeat polymorphism (TRP) and a IS6110 transposition were identified. Typing of all 104 isolates in the IS6110 RFLP cluster with the six DNA polymorphisms endorsed the separate line of transmission established by contact tracing. These findings suggest that the microevolution of M. tuberculosis can be used to resolve separate transmission chains in large outbreak clusters.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 29 GL-5

SURROUNDED BY MYCOBACTERIA

Joseph O. Falkinham, III Department of Biological Sciences Virginia Tech Blacksburg, Virginia 24061-0406 Phone 1-540-231-5931 FAX 1-540-231-9307 E-mail [email protected]

Humans, animals, and plants are surrounded by mycobacteria. The environmental opportunistic mycobacteria (also called nontuberculous or atypical mycobacteria) include over 100 species; many of which cause disease. Infections include cer- vical lymphadenitis in children and pulmonary disease and skin infections in adults. Evidence that the environment was the source of human disease was gained from the identity of DNA fingerprints of mycobacterial isolates from patients and either their household water or potting soils. Recently, a number of reports have documented a dramatic increase in pulmonary disease caused by these mycobacteria amongst elderly and slender men and women who lack all of the classic predisposing risk factors (e.g., smoking, exposure to dusts). Although slowly growing with generation times of one-half to one day, the environmental opportunistic mycobacteria survive, grow, and persist in a number of habitats that are shared with humans and animals. The environmental opportunistic mycobacteria are oligotrophs; able to grow in water containing greater than 50 µg AOC/L. Survival and persistence in the environment is due, in part, to the thick, impermeable, hydrophobic, lipid-rich envelope of mycobacterial cells. Although the hydrophobic wall reduces the rate of transfer or hydrophilic nutrients, it promotes attachment to surfaces where mycobacteria form biofilms. Hydrophobicity also contributes to disinfectant- (e.g., chlorine and biocides) and antibiotic-resistance. For example, mycobacteria enter- ing a water treatment system on particulates survive disinfection and grow during travel in the distribution system in the absence of competitors. Hydrophobicity also promotes the aerosolization of mycobacteria from water to air in environments such as showers and hot tubs in the home and occupations where aerosols are generated.

30 ESM 2009 GL-6

The use of Interferon Gamma Release Assays as an aid in the control of tuberculosis

Jean-Pierre Zellweger Swiss Lung Association, Berne, Switzerland

Interferon Gamma Release Assays (IGRAs) are in vitro tests detecting the presence of latent tuberculosis infection (LTBI) in asymptomatic persons who may have been infected by M. tbc in a recent or remote past and who may benefit from a preventive treatment to decrease the risk of later reactivation of tuberculosis. Basically, the IGRA tests rely on the same immunological phenomenon as the tuberculin skin tests, but they do it in a much more specific way, because the tests are not influenced by a prior vaccination with BGC or by an infection with most of the non-tuberculous mycobacteria present in the environment. Therefore, the indications and the use of the IGRA tests are fundamentally the same as for the tuberculin skin tests : • Detection of LTBI in persons in contact with an index case of tuberculosis • Detection of LTBI in persons with a high risk of tuberculsois, if infected (immunosuppressed patients, patients receiveing or due to receive immunosuppressive therapy, small children) • Surveillance of exposed health care workers (as the test can be repeated without risk of inducing a booster effect) • Aid to the diagnosis of tuberculosis in cases where a bacteriological examination is not feasible or not reliable (severe extrapulmonary TB, TB in children)

In spite of their superiority, the IGRAs are not totally devoid of problems in practice and the best use of them is still a matter of debate. Some Guidelines recommend their use only for the confirmation of positive TST among contacts (the so-called two-step testing procedure) whereas others recommend the routine replacement of the TST by IGRAs. Performing only one test is easier, and avoids a possible influence of a prior TST on the IGRA response. The predictive value of the new IGRAs seems to be superior to the predictive value of TST, reinforcing their usefulness, if the preventive treatment is corectly precribed and followed. One intriguing phenomenon is the possible reversion of a positive IGRA after conversion, possibly indicating that some infected contacts may be able to eradicate the mycobacteria without treatment.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 31 GL-7

A NOVEL DIAGNOSTIC TEST TO DIFFERENTIATE LATENT TB INFECTION AND ACTIVE DISEASE

Lee W. Riley MD, School of Public Health, University of California, Berkeley

It is well recognized that the treatment of latent TB infection (LTBI) is a highly effective TB prevention strategy, which is still not widely practiced in most parts of the world. Most TB-endemic countries rely on BCG vaccine to prevent TB. LTBI treatment requires contact investigation, which is not done in most “BCG countries”. One reason for this reluc- tance to practice contact investigation is the lack of a reliable test that can distinguish LTBI from TB. Thus, a test that can unequivocally distinguish LTBI from TB could alter the current national prevention programs in TB-endemic countries. We have identified a set of M. tuberculosis cell wall proteins that are expressed when the bacilli replicate in vivo, but not when they are in a nonreplicative state. Their continued expression is associated with disease progression in infected mice, and mouse T cells are sensitized as these proteins are continually expressed in vivo. Exploiting this observation, we developed a bioassay that is able to distinguish LTBI from active disease in a mouse model. The assay is based on IFNγ induction by T cells exposed to a set of synthetic peptides based on the cell wall protein called Mcep1A. The Cornell mouse model was used to study the response of spleen cells exposed ex vivo to these peptides. Cells from untreated mice expressed 7-9-fold higher levels of IFNγ than those from treated mice at 24 and 32 weeks of infection, as measured by ELISA. Blood cells from healthy tuberculin skin-test positive, QuantiFERON-negative (n=3) and TST-negative, QuantiFERON-negative (n=3) human volunteers showed no response to the peptides. These peptides are currently under evaluation in newly diagnosed TB patients. If the assay can show a response in these TB patients at levels similar to those observed in diseased mice, this assay can be converted into an immunochromatographic (“dip stick”) format. Such a test then can be used to readily differentiate those with LTBI and active disease, and could then be incorporated as part of National TB Control Programs.

32 ESM 2009 GL-8

NEW LIVE TUBERCULOSIS VACCINE STRATEGIES

Jesus Gonzalo Asensio, Ainhoa Arbues and Carlos Martín Department of Microbiology, University of Zaragoza. Spain http://genmico.unizar.es

BCG, the current vaccine against tuberculosis (TB), has been used for more than 80 years but is ineffective at providing protection against adult pulmonary TB. New tuberculosis vaccine candidates and TB vaccination strategies, conferring better protection against pulmonary tuberculosis than the current vaccine BCG, are needed. In the recent decade, a global pipeline of novel TB candidates has emerged. Pioneering strategies for the development of more effective vaccines today have lead to the discovery of subunit vaccines, which have proved ineffective at providing better protection that BCG in various animal models. Different heterologous prime BCG and boost with subunit strategies are in clinical trials with the aim to improve efficacy of BCG. More recently, clinical trials with recombinant BCG vaccines have started with the aim to find candidates to be used as prime, preventive vaccines. Another innovative strategy, live attenuated Mycbacterium tuberculosis vaccines, in late preclinical investigation, are promising new preventive vaccine candidates to replace BCG. Based upon the observation that phoP is an essential gene for M. tuberculosis virulence, we rationally attenuated the tubercle bacillus by inactivating phoP (Perez et al, Mol Micro 2001). The mutant was shown to be strongly attenuated in cellular and animal models. Moreover, the phoP mutant resulted more attenuated than BCG Pasteur in immunocompromised SCID mice and this vaccine candidate protected guinea pigs and non human primates against tuberculosis infection (Martin et al Vaccine 2006, Verreck et al PLoS ONE 2009). Both, the attenuated phenotype and the protective immunity conferred against tuberculosis infection can be accounted for by the mechanism of action of PhoP, which has been recently shown to be crucial for intricate virulence network of M. tuberculosis (Gonzalo Asensio et al PLoS ONE 2008). This observation was used to construct a new generation of live vaccines based on phoP inactivation carrying a second additional mutation which affects the synthesis of a new family of lipids associated to M. tuberculosis virulence. It is estimated that at least 20 vaccine candidates should enter phase I safety trials with around half going forward for immunological evaluation in phase II trials and leading to four phase III efficacy trials with the goal to reach an effective licensed vaccine in 5-7 years (Young and Dye, Cell 2006). The discovery and use of a new TB vaccine better than BCG is key to reach the 2050 objective of TB eradication.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 33 GL-9

REGULATION OF Mycobacterium tuberculosis CELL WALL LIPID COMPOSITION AND ITS EFFECT ON IN VIVO BACTERIAL PERSISTENCE

Lee W. Riley School of Public Health, University of California, Berkeley

The hallmark of M. tuberculosis is its ability to survive for many years in an infected host to establish latent tuberculosis infection (LTBI). We propose a new model of latent infection that is based on the idea that this organism may simply have readapted its “housekeeping” metabolic function to a new environment for its long-term survival. We propose that M. tuberculosis, which evolutionarily most likely originated in soil, has readapted a soil-survival strategy its ancestral species possessed to the granuloma environment in the human host. Granuloma cells constantly turn over every few days to weeks, and after they die, they undergo replacement by new cells that migrate into the granuloma. Hence, M. tuberculosis needs to readapt to this constantly changing environment, and we provide evidence that this adaptation is mediated by M. tuberculosis remodeling its cell envelope in response to signals produced by dead granuloma cells. This remodeling is mediated by a family of operons called mce (mce1,2,3,4). Disruption of the operons results in profound changes in lipid profile of the cell wall. The mce1 operon mutant causes free mycolic acids (MA) to accumulate on its surface, and other operon mutants (mce2,3,4) show evidence of lipid profile changes in the cell wall. These operon products serve as energy-dependent lipid importers. Lipid products released from dead host granuloma cells that turnover may be used as carbon sources for the resident M. tuberculosis. Thus, the “housekeeping” lipid metabolic function of M. tuberculosis may have been readapted in the granuloma environment as a way for this organism to survive, similar to the way its ancestral saprophytic organism survived in soil by scavenging dead organic materials as carbon sources. Further elucidation of the interaction between M. tuberculosis cell wall and granuloma cell turnover may contribute to a new understanding of the mechanism of LTBI.

34 ESM 2009 GL-10

CDC’S GLOBAL TB LABORATORY ACTIVITIES

Thomas M. Shinnick, Associate Director of Global Laboratory Activities, Division of TB Elimination, Centers for Disease Control and Prevention, 1600 Clifton Road, MS-G35, Atlanta Georgia 30333 USA. email: [email protected]; FAX: 1-404- 639-1287; Tel: 1-404-639-1474

A key bottleneck in health service delivery is weak laboratory capacity. This is particularly true for drug-resistant TB — less than 5% of MDR TB cases are currently being detected globally. To meet the 2015 targets of the Stop TB Global Plan, 60 million culture tests and 5 million drug susceptibility tests will be needed annually. This will require establishing at least 2,000 new culture laboratories and training of more than 20,000 laboratorians. At least US$ 1 billion will be needed annually for building TB laboratory infrastructure and recurring costs. However, the benefit to cost ratio of such investments is estimated to be 9:1 in populations with a high prevalence of HIV infection. Meeting the 2015 goals could save countries in sub-Saharan Africa alone as much as US$ 52 billion annually. While training of bench-level technicians relies on TB-specific expertise, laboratory capacity building relies more on cross-cutting expertise in infrastructure, biosafety, human resource development, supply chain management, logistics, quality assurance programs, management principles, information systems, data management, and accreditation processes. As such, TB laboratory strengthening efforts can build on lessons-learned from the building of laboratory networks for polio, measles, SARS, influenza, HIV/AIDS, and other diseases. The goal of our TB laboratory strengthening efforts is the creation of a network of laboratories that can provide reliable, high quality testing and which is based on quality laboratory management principles and integrated across disease programs, especially HIV and TB. A systems approach is used to optimize laboratory testing and information exchange. The approach involves understanding the structure, performance, and cost of the network; developing referral processes to ensure prompt flow of specimens and information; and using quality-improvement principles to continually evaluate and improve the performance of the network. While TB laboratory strengthening plays the central role in our efforts, the overriding goal is to ensure that a broader health systems approach is used to maximize the impact and sustainability of the investments.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 35 GL-11

DEVELOPMENT AND VALIDATION OF NEW TB DIAGNOSTIC TESTS IN BRAZIL: EXPERIENCE OF REDE-TB

Brazilian Tuberculosis Research Network: A Multi Disciplinary Collaborative Research Project

Introduction In Brazil, which has an estimated 124,000 cases of TB per year, there has been a significant gap in communication and understanding between TB programmatic experts, academics, the community, and nongovernmental organizations. In recognition of this gap, a National TB Research Network was established in 2002 to bring these constituencies together to promote an integrated, multi-disciplinary and multi-institutional strategy for TB control in Brazil. In the last years, Rede- TB has established a solid relationship among the National Tuberculosis Control Program and Oswaldo Cruz Foundation that has helped to foster Brazilian leadership and competency in the development and evaluation under field conditions of new diagnostics for TB.

Objectives To develop new diagnostics, vaccines and drugs for the prevention and cure of TB, including MDR-TB, and develop improved, therapeutic alternatives with the renewal of drugs, new formulations, drug associations, use of drugs already available in the market, and immunotherapy. To perform pre-clinical and clinical studies of new diagnostic tests against TB using adequate ethical standards and to capacitate clinical sites for explanatory and pragmatic trials. To carried out pragmatic clinical trials and cost-effective analysis of alternative interventions for TB control that include diagnostic methods and health service strategies To improve case detection by changing health behaviour and mobilizing communities. To build a successful research partnership model that has every potential to stimulate changes in national standards and practice in ways that serve country needs.

Conclusions Through this approach, it is expected that locally algorithms based on clinical features, antibiotic response, and chest ra- diography will be validated, and clear guidelines issued about which patients might also benefit from mycobacteria culture or new phenotypic / molecular diagnostic techniques. Additionally, the incorporation of new tests into clinical practice will better planned and regulated, and national policy makers with better decision-making models will be provided.

36 ESM 2009 GL-12

EXPERIENCE OF A SUCCESSFUL MYCOBACTERIOLOGY LABORATORY NETWORK IN ESPIRITO SANTO- BRAZIL

Moisés Palaci Universidade Federal do Espírito Santo, Vitória, Brazil

The State of Espírito Santo occupies an area of approximately 6,750 square miles on the coast of Brazil. The economy is mainly based on the production of steel, harborage activity, agriculture, a large number of small industries, and tour- ism. The population of the State is approximately 3.2 million, with the majority living in metropolitan Vitória, the capital, which is located on an island and connected to the mainland by several bridges. The annual incidence of TB on the island of Vitória is approximately 70 cases per 100,000 inhabitants. Each year approximately 1,500 new cases of TB (65% smear positive) are reported for the State of Espírito Santo with 60% occurring in the City of Vitória and its 5 neighboring cities. The Núcleo de Doenças Infecciosas has organized a local network of mycobacteriology laboratories in the Epírito State, Brazil, Five local laboratories have been integrated into this network. This network was established by NDI researchers and is committed to keep a partnership with the City Department of Health of each location and their laboratories. The first phase of these partnerships consisted in reforming and restructuring the laboratories to enable them to accomplish the technical requirements, data processing, and biosafety regulations involved in these projects. To this extent, basic equipment and computers were installed to allow for the maintenance of mycobacterial culture and sharing of data over an Internet database. The second phase consisted of training laboratory staff to properly and safely complete the necessary bench work and data processing procedures. Therefore, a considerable efforts and time has been spent for setting up and to keep this system working. With the establishment of this network, NDI has gained earlier access to TB patients and better conditions to conduct several clinical trials, including IND trials. In addition, capacitating local TB laboratories in the metropolitan region of Vitória to perform mycobacterial cultures, allowed us to increase the detection rate of TB cases at about 24%.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 37 GL-13

SCREENING OF MOLECULES WITH ANTI-TB ACTIVITY, FROM THE BRAZILIAN CERRADO PLANTS, AND SYNTHETIC METALLO-ORGANIC COMPOUNDS

Clarice Leite Universidade Estadual Paulista, Faculdade de Ciências Farmacêuticas

Worldwide, tuberculosis (TB) remains the most frequent and important infectious disease causing morbidity and death. Among all countries in the Americas, Brazil reports the second-highest TB mortality and morbidity, comprising a prevalence of 62/100.000. The global resurgence of TB and the rapid emergence of MDR-TB, underscore the importance of the development of new antituberculous drugs. Plants have provided many drugs in the past, and they remain a rich source of novel compounds. Plant extracts are among the most attractive sources for developing new drugs and have been shown to produce promising results in the treatment of several diseases. Our research group deals in projects that integrate the chemical and anti-TB activity of plants that compose the bioma of the Brazilian Cerrado, a savannah like vegetation. Many of those plants are commonly used as natural remedies by people living in these areas to treat many illnesses. To perform the phytochemical step we used chromatographic techniques, and to determine the structure of the isolated compounds we used mainly spectrometric methods. To evaluate the activity of the extracts, enriched fractions and pure substances against M. tuberculosis we use

the resazurin microtiter assay (REMA) and M. tuberculosis H37Rv ATCC 27294 strain. In total were studied 77 extracts from 39 plants, distributed into 20 families. From all extracts assayed 23% showed promising activity, bellow or equal to 125 µg/mL. The triterpene bassic acid from B. fagifolia showed strong antitubercular activity with MIC values of 2.5 µg/mL comparable to MICs of some first-line tuberculosis drugs. The results indicated that plants of "cerrado" present fractions and compounds with promising anti tuberculosis activity. By the way, the use of natural compounds from plants is problematic, due to difficulty in obtaining pure substances and their low availability. Within the pipeline of new synthetic compounds with potential effectiveness in the treatment of TB, there are 7 novel compounds, which are in various stages of clinical development. Inside this group however, there are complexes that associate metals to organic compounds. Using the thiosemicarbazones, semicarbazones and hidrazones derivates as ligands, we proposed the complexation with Vanadium, to obtain organo-metallic compounds. We determined the anti-M. tuberculosis activity of these compounds using REMA and the study of the citotoxicity of the ligands and complexes was performed using murine macrophage cell line J774. We analyzed 37 compounds (14 free ligands and 23 vanadium complexes) and from of this, 17 (46%) presented promising MIC values varying between 0.97 and 7.80 μg/mL. The vanadium complexes of hydrazones, semicarbazones and tiossemicarbazones derivates showed high antiTB activity, most of the time this activity was increased from 2 to 10 times when compared with the free ligands. However due to high citotoxicity of hydrazones, semicarbazones and tiossemicarbazones derivates, the increase in the activity of the complexes didn’t compensate the citotoxicity of the ligands.

38 ESM 2009 GL-14

CLINICAL TRIALS OF DRUGS AND DIAGNOSTIC TESTS: THE CHALLENGES IN MYCOBACTERIOLOGY

Moisés Palaci Universidade Federal do Espírito Santo, Vitória, Brazil

Tuberculosis (TB) therapy has three major microbiologic goals: (1) initial killing of actively multiplying organisms in order to achieve early control of the disease and reduce infectivity (early bactericidal activity [EBA]); (2) eliminating slowly growing mycobacteria in order to minimize relapses (sterilizing activity); and (3) preventing the emergence of drug resistance (1). Currently, two month sputum culture conversion on solid medium is the best established predictor of treatment outcome. Early bactericidal activity (EBA), determined by the serial decline in sputum M. tuberculosis colony counts (CFU), is a commonly used tool for comparing new drugs to current anti-TB drugs and dose finding. EBA has been measured as the rate of decrease in colony counts of mycobacteria in quantitative sputum cultures obtained during the first days of therapy. Measurement of EBA is intended to be a rapid means of assessing the relative potency of new drugs during early treatment. The development of new drugs for TB treatment has been hampered by the lack of an early surrogate marker that reflects long term non-relapsing cure. Measurement of EBA by quantitative culture however is time consuming and labor intensive. The ideal marker would measure events early during treatment and be accurate regard- less of the drug action or regimen being tested. In this lecture we will explore the potential role and the main limitation of surrogate markers for TB treatment.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 39 GL-15

NEW, EASY-TO-USE TOOLS FOR QUALITY-CONTROLLED GENOTYPING OF M. tuberculosis COMPLEX STRAINS

Caroline Allix-Béguec1,2,3, Christine Hubans3, Stéphanie Ferreira3, and Philip Supply1,2,3 1 - INSERM U629 2 - Institut Pasteur de Lille, Lille 3 - Genoscreen, Lille, France, France

Mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) typing has become a major method for fast and high-resolution genotyping of Mycobacterium tuberculosis complex isolates. A system based on 24 loci has been proposed for international standardization. Several population-based studies have been published, showing similar predictive value of this method compared to the previous gold standard IS6110 RFLP for studying tuberculosis transmission in Western European settings. As a result, this method is being internationally adopted, often in combination with spoligotyping, as the new standard method for TB molecular epidemiology. New, easy-to-use tools and options have recently become available, which facilitate quality-controlled use of this technique and interpretation of the results obtained. MIRU-VNTR typing services are already used by international Reference Centers and laboratories, for out- sourcing their genotyping (including of M. bovis strains) and/or for QA/QC evaluation. Quality-controlled MIRU-VNTR calibration, validation and typing kits, as well as on-site trainings greatly facilitate standardized set up and efficient use of MIRU-VNTR typing in user’s laboratory. Bioinformatic tools, including MIRU-VNTR Data Manager, have been developed for further automating and streamlining the genotyping process, as well as ensuring direct compatibility with MIRU- VNTRPlus Database for data interpretation. We hope that the availability of these tools for easier and more efficient real-time genotyping will contribute to improve molecular-guided TB control and surveillance.

40 ESM 2009 Abstracts of ORAL PRESENTATIONS (OP)

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 41

OP-1

Mass Spectrometry for Molecular Typing of the Mycobacterium tuberculosis Complex: One Platform and Multiple Assay Formats

C. Honisch1, M. Mosko1, C. Arnold2, S. Gharbia2, S. Feuerriegel3, S. Niemann3 1 - SEQUENOM, Inc., San Diego 2 - Health Protection Agency, London 3 - Molecular Mycobacteriology, NRC for Mycobacteria, Forschungszentrum Borstel, Borstel

Objectives The analysis of nucleic acids by mass spectrometry has evolved to a user friendly technology for characterizing DNA, and RNA via SNP genotyping and comparative sequencing in clinical research, agricultural applications, molecular medicine and non-invasive prenatal diagnostics research. Recently, the technology has become a versatile tool for microbial detection and identification utilizing comparative sequence analysis. An example is the successful application to 16S based typing of mycobacteria. Here, we adopted the technology to perform high throughput spoligotyping and detection of resistance conferring SNPs.

Methods Assays were designed in silico for spoligotyping analysis and detection of key resistance mutations. Both assays were evaluated by using well characterized reference collections.

Results For MassARRAY 43 spacer oligonucleotide probes were designed and grouped into two multiplexed assays (TypePLEXTM). Over 200 characterized strains from different reference centers representing the major M. tuberculosis complex lineages were analyzed by the MassARRAY spoligotyping assays. Results were in concordance with classical spoligotyping data. For detection of resistance mutations, assay were developed based on the MassCLEAVETM system. Resistance regions are amplified by PCR with a tagged primer system followed by in vitro transcription of both DNA strands. Subsequent endonuclease digests of the RNA transcripts at the bases cytosine and uracil result in four mixtures of RNA cleavage products. Resistance is identified by correlating acquired spectra with theoretical peak patterns predicted for in silico cleavages of sequences contained in a reference database. The first assays have been successfully evaluated in a set of reference strains, further analyses are in progress.

Conclusion Mass spectrometry specific assay formats for genotyping and comparative sequence analysis generate highly accurate qualitative and quantitative data and provide a toolbox for molecular typing of microbes and viruses. Existing typing schemes can be translated onto the mass spectrometry platform and new typing schemes can easily be developed.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 43 OP-2

Clustering of spoligo-patterns: towards an automation of Mycobacterium tuberculosis complex classification

Borile C 1, Refrégier G 2, Labarre M 1, Franz S 1, Mézard M 1, Sola C 2 1 - LPTMS, bât. 100, Université Paris-Sud, Centre scientifique d’Orsay, 15 rue Georges Clémenceau, 91405 Orsay cedex 2 - IGEPE bât. 400, Université Paris-Sud, Centre scientifique d’Orsay, rue Gregor Mendel, 91405 Orsay cedex

Spoligotyping is a typing method detecting the presence or absence of specific regions called spacers. These spacers are grouped on what is called the DR locus (Direct Repeat locus) that belongs to the CRISPR locus family (Clustered Regularly Interspaced Palindromic Repeats). The DR locus in Mycobacterium tuberculosis complex is believed to evolve solely by deletion. Using the spoligotyping technique, specific families of Mycobacterium tuberculosis complex strains have been recognized, showing that the DR locus is phylogenetically informative. Specific signatures are recognized by experts so that each spoligo-pattern is easily assigned to a specific family. Amazingly however, when using all available softwares for clustering the data, the strains of a specific family are not always clustered in the same groupe using various methods. We implemented a new algorithm to cluster these data. Until now, the single publicly available software to achieve this task, SpotClust, provided only sub-optimal results. Our system is based on an evolutionary model taking into account that spacers can be deleted as a large group. This is not the case when using the Jaccard distance in the commonly used Bionumerics software, that mimicks a one-by-one spacer deletion process. We will present data showing under what conditions this algorithm gives significantly better results than the commonly used one. This studies leads toward an automation of Mycobacterium tuberculosis complex classification, an otherwise time consuming and sometimes debatable topic.

44 ESM 2009 OP-3

IDENTIFICATION OF THE INSERTION ELEMENT IS6110 IN PHOP PROMOTER IN A HIGH TRANSMISSION Mycobacterium tuberculosis STRAIN: A CLUE TO PHENOTYPIC VARIATION

Andrea Sandoval1,2, Andrés Cubillos1,2, Alejandro Reyes3, Nidia Correa2,4, Jaime Robledo2,4, Maria Mercedes Zambrano1, and Patricia Del Portillo1,2 1 - Corporación CorpoGen, Bogotá, Colombia. 2 - Centro Colombiano de Investigación en Tuberculosis CCITB, Bogotá, Colombia. 3 - Center for Genome Sciences, Washington University School of Medicine, St. Louis, Missouri, USA 4 - Corporación para Investigaciones Biológicas CIB, Medellín, Colombia

Aim The insertion element IS6110 can mediate genetic diversity in Mycobacterium tuberculosis (MTB) strains due to its capacity to move and cause rearrangements, deletions and insertions. Transposition of these elements can therefore affect gene expression and alter the phenotype of MTB. In this study we analyzed the insertion sites of IS6110 in two clinical MTB Haarlem genotype strains that present differences in transmissibility as demonstrated in a cohort of patients and household contacts from Colombia.

Methods Restriction Fragment Length Polymorphism (IS6110-RFLP) was performed and revealed genomic differences between the two strains. DNA was isolated, digested with XmaI and ligated to specific adapters designed for this purpose. Ligation Mediated PCR (LM-PCR) was carried out to amplify the regions flanking IS6110 insertion sites. A library containing the amplified products was constructed and sequenced clones were mapped against the annotated sequenced genomes. PCR was used to confirm the sites of insertion.

Results Twelve different insertions were identified; nine were common to both strains (Rv2336, Rv1754c, Rv0963c, Rv0403c, Rv1358, Rv2813/DR, Rv2254c, Rv0795 and PPE34), two were specific for the high transmission strain (DR region and transcriptional regulator phoP), and one was found just in the low transmission strain (PPE46).

Conclusions LM-PCR allowed us to identify IS6110 flanking regions and localized the genomic differences between two strains with contrasting transmission pattern. Most of the insertions occur in conserved hypothetical proteins, followed by proteins involved in cell wall synthesis and cell processes, regulatory proteins and members of the PE/PPE family. The DR region, considered a hotspot for insertions, had three insertions, two common to both strains and one in the high transmission strain. Since the phoP gene regulates several functions implicated in virulence, it is possible that the IS6110 insertion in the phoP promoter could enhance transmission by acting as a portable promoter and inducing gene expression, as has been reported before in M. bovis.

Acknowledgment Consorcio Colombiano de Investigación en Tuberculosis, CCITB. 4312004

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 45 OP-4

GENOMIC INTERROGATION OF Mycobacterium tuberculosis ISOLATES FROM BRAZIL

Oelemann, Maranibia C 1, Gomes, Harrison M 1, Willery, Eve 2, Lima, Karla Valéria B 3, Possuelo, Lia 4, Locht, Camille 5, Goguet de la Salmonière, Yves-Olivier L 6, Gutierrez, Maria Cristina 6, Supply, Philip 7, Suffys, Philip N 1 1 - Laboratory of Molecular Biology Applied to Mycobacteria, Oswaldo Cruz Institute, Rio de Janeiro, Brazil 2 - Laboratory of Molecular Mechanisms of Pathogenesis of Respiratory Pathogens, INSERM U629 and Institut Pasteur de Lille, France 3 - Evandro Chagas Institute, Belém, Brazil 4 - Center of Scientific and Technological Development, Porto Alegre, Brazil 5 - Laboratory of Molecular Mechanisms of Pathogenesis of Respiratory Pathogens, INSERM U629 and Institut Pasteur de Lille, France 6 - Department of Infection and Epidemiology, Institut Pasteur, Paris, France 7 - Laboratory of Molecular Mechanisms of Pathogenesis of Respiratory Pathogens, INSERM U629 and Institut Pasteur de Lille, France 1 - Laboratory of Molecular Biology Applied to Mycobacteria, Oswaldo Cruz Institute, Rio de Janeiro, Brazil

Background The Latin-American Mediterranean (LAM) spoligotype“clade” is among the six major M. tuberculosis spoligotype families and is particularly prevalent in SouthAmerica. In certain regions, there is a dominance of geographically specific and genetically homogeneous strain lineages. Here, we have studied the genetic diversity and the consistency of the LAM and other families, by analyzing Mtb isolates from three Brazilian regions including Rio de Janeiro (South East), Belém (North), and Rio Grande do Sul (South).

Methods and Findings A PCR-based standardized genotyping system, based on amplification of 15 to 24 mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) loci combined with spoligotyping, has been shown to be proficient for molecular-guided evaluation of TB transmission. We tested the applicability of this system for molecular epidemiological analysis of 369 M. tuberculosis isolates from three regions of Brazil. Deligotyping, targeting multiple large sequence polymorphisms (LSPs), and MIRU-VNTRplus identification database were additionally used to confirm phylogenetic identification. The high congruence between the different typing results showed the countrywide supremacy of the Latin-American-Mediterranean (LAM) lineage, comprised of three main branches. Nevertheless, by distinguishing 321 genotypes among the 369 isolates, combined MIRU-VNTR typing and spoligotyping demonstrated the presence of multiple distinct clones. Noteworthy, 27 of the 32 clusters identified were exclusively composed of patient isolates from a same city, consistent with expected patterns of local TB transmission.

Conclusions Notwithstanding the challenges, high-capacity mycobacterial genotyping may now become a usable tool to guide TB control efforts, at least on sentinel sites or targeted risk-populations in high TB burden countries. The interrogation of large sequence polymorphisms (LSPs) revealed that certain lineages or clones present genomic features that could change the phenotype and play a role in the clinical properties of specific Mtb strains. This is the first countrywide report of an in-depth analysis on the genomic diversity of M. tuberculosis isolates from Brazil.

Acknowledgments Fiocruz, CAPES, CNPq, FAPERJ, ICOHRTA, NIH, INSERM and Institut Pasteur.

46 ESM 2009 OP-5

PENITENTIARY POPULATION OF Mycobacterium tuberculosis IN KYRGYZSTAN: EXCEPTIONALLY HIGH PREVALENCE OF THE BEIJING GENOTYPE AND ITS RUSSIA-SPECIFIC SUBTYPE

Igor Mokrousov 1, 2, Violeta Valcheva 1, 3, Nurmira Sovhozova 4, Almaz Aldashev 4, Nalin Rastogi 1, Jainagul Isakova 4 1 - Institut Pasteur de Guadeloupe, France 2 - St. Petersburg Pasteur Institute, St. Petersburg, Russia 3 - Institute of Microbiology, Sofia, Bulgaria 4 - Institute of Molecular Biology and Medicine, Bishkek, Kyrgyz Republic

Objective To identify genotypes and drug resistance properties of M. tuberculosis isolates from Kyrgyzstan’s prison inmates, a population with high risk for TB; to compare in regional and global context.

Methods 56 M. tuberculosis DNA samples from sputum of HIV-negative Kyrgyz prison inmates, 2008, were typed by spoligotyping, VNTR (12 MIRU and 3 hypervariable [HV] loci), IS6110-inverse-PCR, LAM-PCR. rpoB and katG mutations were detected using TB-Biochip kit.

Results Beijing genotype was detected in 42 of 56 samples. 12-locus MIRU-VNTR typing showed 8 of 56 samples to be mixed cases; 7 of them contained a Beijing strain. MIRU analysis demonstrated a high homogeneity of the studied collection (HGI=0.66) while 28 of 56 strains had a profile 223325153533 corresponding to Beijing/M2 subtype highly prevalent in different Russian settings (Mokrousov, 2004, 2008). Four Beijing strains belonged to types M33 and M70 specific for East Asia. Regarding non-Beijing variants, a comparison of their spoligoprofiles with SITVIT2 database (Institut Pasteur de Guadeloupe) revealed a presence of minor global and Eurasia (Europe/Russia) specific types SIT262/Haarlem, SIT73, SIT254/LAM found in ex-USSR and Europe but very rare in East Asia and global type SIT42/LAM that is also prevalent in different parts in Eurasia. Three hypervariable loci, QUB-3232, VNTR-3820 and VNTR-4120, permitted to subdivide 28 Beijing strains with MIRU12 profile 223325153533 into 11 subtypes shared by 1 to 9 strains. RIF and INH resistance was detected in 28% and 55% samples. 13 of 15 MDR strains belonged to Beijing genotype. Comparison of the rate of drug resistance mutations in different Beijing subclusters revealed no statistically significant difference.

Conclusions The penitentiary population of M. tuberculosis in Kyrgyzstan shows a strong affinity to the north-west Eurasia, especially, Russia, and a weak relatedness to East Asia. Beijing genotype constituted 75% of the entire collection while half of the studied strains belonged to the Beijing/M2 MIRU-defined subtype that is the major Beijing variant in Russia. MDR-TB was detected in 27% samples that is similar to the Kyrgyzstan’s civilian population. IS6110-inverse PCR and HV-VNTR loci were shown to be useful for detection of and subtyping within the Beijing genotype, directly in sputum-extracted DNA.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 47 OP-6

THE UNIQUE ENDEMIC NATURE OF BEIJING GENOTYPE STRAINS IN OKINAWA, RYUKYU ISLANDS OF JAPAN AS REVEALED BY NEWLY DESCRIBED 15 AND 24-LOCI MIRU-VNTR TYPING SCHEMES

Julie Millet, 1 Chika Miyagi-Shiohira, 2 Nobuhisa Yamane, 2 Igor Mokrousov, 1,3 Nalin Rastogi 1 1 - Unité de la Tuberculose et des Mycobactéries, Institut Pasteur de Guadeloupe, Abymes, Guadeloupe 2 - Department of Laboratory Medicine, Graduate School and Faculty of Medicine, University of the Ryukyus, Okinawa, Japan 3 - St. Petersburg Pasteur Institute, St. Petersburg, Russia

Tuberculosis (TB) in the eastern Asiatic countries is mainly caused by strains of Mycobacterium tuberculosis belonging to the Beijing lineage which was identified back in early nineties using IS6110-RFLP and spoligotyping. Associated with multiple drug-resistance (MDRTB), this highly homogeneous genogroup is characterized by little molecular diversity. Several studies have recently emphasized the utility of using minisatellites in conjunction with IS6110-RFLP or spoligotyping for a better discrimination of Beijing strains. In a recent study, we genotyped Beijing TB strains collected in Okinawa (Ryukyu Islands, Japan), by using a discriminative selection of 8 MIRU loci (chosen from the classical 12-loci MIRUs), and 7 QUB markers. This typing scheme excluded 4 MIRU loci (MIRU2, 4, 20, and 24), that were found to have a too low discriminatory power within an in-house Beijing database containing 694 strains (Millet et al., J. Clin. Microbiol. 2007, 45:3606–3615). In the present study we evaluated the full “classical” 12-loci typing as compared to the newly described 15-loci and 24- loci MIRU-VNTR typing schemes, which include 9 and 12 new MIRU loci respectively. We compared the results obtained in Okinawa (an insular setting; n=72) with those recently published for Osaka (n=174 strains) and Kobe (n=175) (Wada et al., FEMS Microbiol Lett. 2009, 291:35-43). A higher discriminatory power of 15-loci versus 12-loci format was seen through percentage of clustered isolates; clustering for 12-loci format in Osaka, Kobe, and Okinawa was 78.3, 81.7, and 68.1% respectively, as compared to 55.7, 48.0, and 37.1% using 15-loci format. Corresponding discriminatory index (HGI) in Osaka, Kobe, Okinawa were 0.901, 0.936, and 0.944 respectively for 12-loci format, as compared to 0.989, 0.989, and 0.992 for 15-loci format. A finer comparison of 15-loci patterns in the 3 settings revealed that contrary to Kobe and Osaka which shared together a high number of similar patterns (25/114 and 25/107 respectively), Okinawa shared a single pattern out of 55 with Kobe, and none with Osaka. The full 24-loci format results further reduced the clustering observed (from 37.1% to 20%, HGI 0.996). The results analyzed by drawing a minimum spanning tree underlined the unique endemic nature of the Beijing genotype strains in the insular setting of Okinawa, and suggest a local evolution of M. tuberculosis Beijing genotype in this island starting from a common pool in mainland Japan.

48 ESM 2009 op-7

Mycobacterium tuberculosis genetic diversity in South Korea

Isdore Chola Shamputa1, Jongseok Lee2, Caroline Allix-Béguec3, Eun-Jin Cho2, Ji-im Lee2, Jin Hong Min4, Lisa C. Goldfeder1, Jin Hee Kim4, Hyung Seok Kang4, Soo Hee Hwang4, Seok Yong Eum2 ,Hyeyoung Lee5, Seung Kyu Park2,4, Philip Supply3,6, Sang Nae Cho7, Laura E. Via1, Clifton E. Barry III1 1 - Tuberculosis Research Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 2 - International Tuberculosis Research Center, Masan South Korea 3 - Genoscreen, Lille, France 4 - Masan National Tuberculosis Hospital, Masan, South Korea 5 - Department of Biomedical Laboratory Sciences, Yonsei University, Wonju, South Korea 6 - Centre National de la Recherche Scientifique, Institut Pasteur de Lille/Institut de Biologie de Lille, Lille France 7 - Department of Microbiology, Yonsei University College of Medicine, South Korea

South Korea has recorded a nine fold decrease in the incidence of TB in the last four decades, however challenges of TB control remain significant as the Republic of Korea is among the 30 countries with the highest numbers of estimated MDR-TB cases. Genotypic analysis of M. tuberculosis has greatly contributed to the control of TB by providing information on transmission dynamics, assessing clonal distribution and expansion of the tubercle bacilli, in investigating of outbreaks and pseudo-outbreaks, and in identifying laboratory cross contamination. However, there is limited information on the molecular epidemiology of TB in South Korea. Genetic diversity of 208 M. tuberculosis isolates from subjects enrolled in a prospective observational cohort study at National Masan Tuberculosis Hospital in South Korea was determined using spoligotyping, IS6110-RFLP and standardised MIRU-VNTR typing based on 24 loci. MIRU-VNTR analysis was performed independently and blindly from spoligotyping and IS6110-RFLP results. Analysis of MIRU-VNTR typing results use in conjunction with MIRU-VNTRplus database predicted that 202 (97.1%) isolates belonged to the Beijing genotype. This prediction was fully confirmed by spoligotyping. Congruence analysis indicated the prevalence of 3 branches among Beijing strains respectively named Korea, Masan and China. Preliminary analysis did not show differential distribution of resistant, MDR or XDR strains among the 3 branches. MDR or XDR isolates were detected in at least 4 clusters concordantly identified by the 3 genotyping methods. Using MIRU-VNTR typing and spoligotyping, 23 clusters of 66 isolates were detected indicating a relatively large diversity of circulating strains despite the prevalence of the Beijing lineage. Standardised MIRU-VNTR typing appeared efficient as a first line discriminatory method for this country with high prevalence of Beijing strains. However, preliminary analyses suggest that clustered cases may not be epidemiologically linked and rather correspond to endemic strains circulating in South Korea.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 49 OP-8

CORRELATION OF MOLECULAR RESISTANCE MECHANISMS AND PHENOTYPIC RESISTANCE TO FIRST-LINE DRUGS IN Mycobacterium tuberculosis STRAINS FROM SIERRA LEONE

Silke Feuerriegel1, Susanne Homolka1, Erik Post2, Barbara Oberhauser2, Abu Garawani George3, Lars Westman3, Foday Dafae4, Sabine Rüsch-Gerdes1, Stefan Niemann1 1 - Research Center Borstel, National Reference Center for Mycobacteria, Parkallee 18, 23845 Borstel, Germany 2 - German Leprosy and TB Relief Association, Würzburg, Germany 3 - National Leprosy/TB Reference Laboratory, Freetown, Sierra Leone 4 - National Program Manager for Tuberculosis and Leprosy, Freetown, Sierra Leone

Background Resistance to first-line drugs (INH, RMP, SM, EMB and PZA) displays a serious problem for the treatment of Mycobacterium tuberculosis infections. Resulting MDR-tuberculosis (resistance to at least INH and RMP) implies an enormous threat for tuberculosis control worldwide. It is therefore of great importance to analyze the genetic basis of clinical resistance, especially in high incidence settings and to correlate molecular resistance data with phenotypic resistance data.

Methods A total of 97 M. tuberculosis strains from previously treated patients in Sierra Leone which displayed resistance to INH (n=32), RMP (n=16), SM (n=39), EMB (n=15) and PZA (n=10), respectively, were sequenced concerning the predominant resistance determining regions (katG, rpoB, rrs, rpsL embB and pncA). Strains resistant to INH with no mutation in katG were also sequenced in the promoter regions of inhA and ahpC. From all strains analyzed 11 showed resistance to INH and RMP and were therefore MDR-TB. Drug susceptibility testing was done by using the proportion method on Löwenstein-Jensen medium.

Results Among INH resistant strains the most common mutation detected is katG315 (65.6%). From all 32 resistant strains 3 had mutations in the promoter regions of inhA and ahpC. Among RMP resistant strains 50% displayed mutations at rpoB531. Sensitivity and specificity of the DNA sequencing of katG and rpoB for detection of INH and RMP resistance were about 90%. Concerning SM resistance none of the resistant strains showed any mutation in rrs, but 46.2 % had rpsL mutations, either at codon 43 or 88. Among EMB resistant strains 46.7% showed mutations at embB306 and 13.3% at codon 332 and 497 respectively. Strains resistant to PZA displayed a number of different mutations throughout the pncA gene. Specificities of sequencing of rpsL, embB and pncA for detection of the respective resistance phenotypes were high (96-100%), whereas sensitivities were lower (46% for sequencing of rpsL, 60% for embB, 70% for pncA).

Conclusion There is a close correlation between data from molecular and phenotypic resistance testing for the determination of INH and RMP resistance in strains from Sierra Leone. Sensitivities of sequencing of resistance determining genes for SM, EMB and PZA resistance were low due to so far unknown resistance determining regions. Thus it is of great importance to gather information on further mechanisms leading to drug resistant MTB strains in different settings.

50 ESM 2009 OP-9

LAM AND HIV: CORRELATION OR CO-INCIDENCE?

McNerney, Ruth, Mallard, Kim London School of Hygiene & Tropical Medicine

The deadly synergy between TB and HIV presents a serious challenge to heath and development. Three decades after the onset of the AIDS pandemic the region with the highest prevalence of HIV infection is sub-Saharan Africa, followed by the Caribbean and Latin America. Molecular typing methods permit differentiation of M. tuberculosis into strain families or genotypes. We have undertaken analysis of genotyping data to compare the prevalence of spoligotype lineage with that of HIV infection. Data was taken from the peer reviewed literature, surveys testing only drug resistant strains or those not fully describing the genotype population were excluded. The Latin-American-Mediterranean (LAM) lineage are identified as typically lacking spoligotype spacers corresponding to oligonucleotides 21 to 24 and 33 to 36. They have been observed in many geographic locations and include the F11 family reported in South Africa. Our analysis suggests that LAM strains are more frequently found in regions with a high prevalence of HIV. Conversely, they are rare in settings where HIV has yet to emerge as a major threat to public health. Interestingly LAM genotypes appear less frequent in African countries such as Uganda, Tanzania and Cameroon which have lower HIV prevalence’s than countries such as Malawi, Zimbabwe and South Africa where HIV rates are in excess of 10%. Why tuberculosis of this genotype should be linked to the HIV epidemic remains a matter of speculation. Although socioeconomic and political factors play a significant role they do not explain the uneven distribution of HIV in sub Saharan Africa. It is now evident that there is diversity in the interaction of M. tuberculosis with its host and strains of differing genotype appear to elicit subtle but significant differences in immune response. We present the hypothesis that strains of tuberculosis belonging to the LAM genotype are associated with enhanced transmission of TB in immunosuppressed populations.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 51 OP-10

Mycobacterium celatum: AN EMERGING PATHOGEN IN THE IMMUNOCOMPETENT. A CASE REPORT

Lyberopoulos Panagiotis 1, Frangopoulos, Fragiskos1, Kontos, Fanourios 2, Zerva Loukia 2, Malagari Aikaterini 3, Papiris Spyridon 1 1 - 2nd Department of Pulmonary Medicine, Attikon University Hospital, Athens, Greece 2 - Department of Clinical Microbiology, “Attikon” University Hospital, Athens, Greece 3 - 2nd Department of Radiology, Attikon University Hospital, Athens, Greece

Mycobacterium celatum is a pathogen for immunocompromised patients but there is little evidence of its pathogenicity among immunocompetent individuals. We report the isolation of M. celatum from a middle-aged immunocompetent woman with bronchiectasis that was initially considered a non-significant finding, but eventually proved extremely dangerous. A 55-year-old Caucasian female never smoker, was referred to our hospital complaining of productive cough over the preceding two weeks accompanied by general malaise. She lived in an urban area and had no history of alcoholism, use of steroids or immunosuppressive drugs. A high resolution computed tomography (HRCT) of the chest showed ill- defined peribronchial densities contained within the right upper lobe with air-bronchograms and branching pattern with a few opacities of tree-in bud formation. There was no lymphadenopathy or pleural effusion. Smears of five sputum specimens (before any treatment) were negative for acid-fast bacilli. These specimens were processed with the NALC/NaOH method and cultured in the BACTEC MGIT 960 system (Becton Dickinson, USA) and on Löwenstein-Jensen medium (BioMerieux, France). Two mycobacterial strains were recovered from these cultures and both strains were identified as M. celatum with the use of the Genotype Mycobacterium CM and AS assays (Hain- Lifescience). PCR restriction analysis (PRA) of a 439-bp fragment of the hsp65 gene and sequencing of the 16S rRNA gene confirmed that they were M. celatum type 1. From the five aforementioned sputum specimens Pseudomonas aeruginosa and Serratia liquefaciens were also isolated and were both sensitive to ciprofloxacin. Ciprofloxacin (1gr/day, per-os) plus low dose azithromycin (500mg twice a week) were administered. Two months later, a control chest HRCT scan showed a considerable improvement; no pathogens were isolated from consecutive sputum samples. Eight months later the patient complained for general malaise, night sweats and cough with blood tinged sputum. Imaging studies revealed a lung apical cavity. Two bronchial washing and two sputum cultures were smear positives and M. celatum was recovered from all. In conclusion, when the American Thoracic Society criteria for the diagnosis of NTM infection are met and M. celatum is identified, it should be considered as the pathogen causing the pulmonary infection even in patients with apparently normal cellular immunity.

52 ESM 2009 OP-11

Mycobacterium avium SUBSPECIES STRAINS FROM HUMAN AND ANIMAL ORIGIN

Radomski, Nicolas 1, Thibault, Virginie 2, Karoui, Claudine 1, De Cruz, Krystel 1, Cochard, Thierry 1, Gutiérrez, Cristina 3, Supply, Philip 3, Biet, Frank 2, Boschiroli, María Laura 1 1 - AFSSA-LERPAZ, Maisons Alfort 2 - INRA-UR1282, Nouzilly 3 - INSERM U629-Institut Pasteur, Lille

The Mycobacterium avium sbsp. avium and Mycobacterium avium sbsp. hominissuis are pathogenic emergent bacterial species belonging to the Mycobacterium avium complex (MAC). These two subspecies can infect and lead to disease to numerous animal species: birds, pigs, cattle, deer, sheep, goats, horses, cats, dogs, etc. Moreover, Mycobacterium avium subspecies have been isolated in HIV infected patients and in immuno-competent patients with pulmonary pathologies. MAC is an ubiquitous bacterial group that can be found in water, in the environment, or even in food. A molecular typing study was undertaken with the primary goal of improving the taxonomic and epidemiological knowledge of MAC. Different strains of Mycobacterium avium sbsp. avium, Mycobacterium avium sbsp. hominissuis, and also of Mycobacterium avium sbsp. silvaticum, isolated from animals, humans, and the environment, were typed by two methods: the Restriction Fragments Length Polymorphism on insertion sequences IS1311 (RFLP1311), which is a standard method to characterise MAC, and the Variable Number of Tandem Repeats-Mycobacterial Interspersed Repetitive Units (VNTR-MIRUs) characterisation, which has been recently developed on Mycobacterium avium sbsp. paratuberculosis. Our results demonstrate that the discrimination power of both methods is comparable (DI of more than 0.92). Therefore, VNTR-MIRUs seems a much better typing method since it is a PCR based method that requires little genetic material for being performed, which is easy to standardize in any laboratory and because the deduced numerical patterns do not require special softwares for being compared in an inter-laboratories fashion.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 53 OP-12

THE CHARACTERIZATION OF MYCOBACTERIA FROM AN OUTBREAK SUGGESTS A REVISION OF THE TAXONOMIC STATUS OF MEMBERS OF THE Mycobacterium chelonae-abscessus GROUP

Sylvia Cardoso Leão1, Enrico Tortoli2, Cristina Viana-Niero1, Suely Yoko Mizuka Ueki3, Karla Valeria Batista Lima4, Maria Luiza Lopes4, Jesus Yubero5 María Carmen Menendez5, Maria Jesus Garcia5 1 - Universidade Federal de São Paulo, São Paulo, Brazil 2 - Centro Regionale di Riferimento per la Diagnostica dei Micobatteri, Ospedale di Careggi, Firenze, Italy 3 - Instituto Adolfo Lutz, São Paulo, Brazil 4 - Instituto Evandro Chagas, Belém, Brazil 5 - Universidad Autonoma de Madrid, Madrid, Spain

An outbreak of infections by rapidly growing mycobacteria (RGM) related to invasive procedures has been ongoing in Brazil since 2004. Isolates from patients submitted to laparoscopic or plastic surgery and to mesotherapy, a cosmetic procedure, were previously identified by molecular methods as Mycobacterium massiliense and M. bolletii, respectively. The similarity of rpoB and hsp65 sequences from the clinical isolates and the corresponding sequences from both M. massiliense and M. bolletii type strains were above the accepted limit for interspecies variability, leading to conflicting results. Therefore, an extensive characterization was carried out with six Brazilian clinical isolates from this outbreak study and type strains from the members of the M. abscessus-M. chelonae group – M. abscessus, M. chelonae, M. immunogenum, M. massiliense and M. bolletii. Phenotypic identification was performed by biochemical tests, high performance liquid chromatography (HPLC) and drug susceptibility testing. Molecular identification included PCR-restriction enzyme pattern analysis (PRA) of the hsp65 gene, as well as rpoB and hsp65 gene sequencing and analysis of the corresponding phylogenetic trees. DNA-DNA hybridization (DDH) and restriction fragment length polymorphism (RFLP) of the 16S rRNA gene were used as the gold standards for RGM speciation. The clinical isolates and the M. abscessus, M. massiliense and M. bolletii type strains could not be separated by phenotypic tests and were grouped in the phylogenetic trees obtained. Also, DDH results confirmed >70% relatedness, and indistinguishable RFLP 16S rRNA patterns were obtained. On the contrary, separation from M. chelonae and M. immunogenum was supported by results from PRA-hsp65, rpoB and hsp65 phylogenetic trees, DDH and RFLP-16S. Taken together, these results led to the proposition that M. abscesus, M. massiliense and M. bolletii represent a single species, that of M. abscessus. Two subspecies are also proposed, M. abscessus subsp. abscessus and M. abscessus subsp. massiliense which can be distinguished by two different PRA-hsp65 patterns, differing in a single Hae III band, and by differences in rpoB (3.4%) and hsp65 (1.3%) sequences.

54 ESM 2009 OP-13

THE SUNNY SIDE OF MYCOBACTERIA

Santos, Ricardo 1, Marques, Marco 2, Oliveira, Pedro 2, Carvalho, Filipe 2, Carvalho, Carla 2, Monteiro, Gabriel 2, Cabral, Joaquim 2, Frade, Raquel 2, Silva, Maria 3, Fernandes, Pedro 4 1 - Laboratório de Análises 2 - IBB-CEBQ-IST 3 - Faculdade de Farmacia, Univ. Coimbra 4 - IBB-CEBQ-IST

Mycobacteria are typically seen as cause of morbidity and mortality worldwide. There is however a Jekyll side to these acid-fast bugs. Taking advantage of some key metabolic pathways, as well as of the particular nature of the hydrophobic cell wall, that enables operation in aggressive, non-conventional environments, non-pathogenic mycobacteria can be used for the production of compounds with application in the pharmaceutical, food and environmental areas. The present work aims to illustrate this concept, by providing examples of the application of non-pathogenic mycobacteria for the production of steroid and siderophore molecules, and for the pinpoint modification of carbocycles. The assessment of the required biocatalytic activity and the early stages of process characterization have been carried out in miniatur- ized bioreactors, allowing for a high level of parallelization, hence providing a high throughput platform for bioprocess development. Going into detail, Mycobacterium sp. NRRL B-3805 was shown to effectively yield androstenedione (AD), a key intermediate in the production of therapeutic steroids, from phytosterols, recovered from residues of the paper industry, while operating in phthalate or liquid silicone environments, an approach that enhances process productivity. Furthermore, this particular strain was shown to also yield the AD out of several polyhydroxylated steroids. Relying on the high-throughput methodology, a library of non-pathogenic Mycobacterium spp. siderophore producers was developed in-house. Scaling-up the production process to bench bioreactor using the most promising strains is envisaged. The same high-throughput methodology has been recently used to screen non-pathogenic mycobacteria for specific catalytic activity, namely hydroxylation, on carbocyclic molecules, with promising results.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 55 OP-14

FAST IDENTIFICATION OF Mycobacterium tuberculosis IN SPUTUM AND CULTURES BASED ON THERMALLY-ASSISTED HYDROLYSIS AND METHYLATION BY GAS CHROMATOGRAPHY-MASS SPECTROMETRY

Erwin Kaal 1,2*, Arend Kolk 3, Sjoukje Kuijper 3, Hans-Gerd Janssen 1,4 1 - Polymer-Analysis group, Van ’t Hoff Institute for Molecular Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands. 2 - ATAS GL International, P.O. Box 17, 5500 AA Veldhoven, The Netherlands. 3 - KIT Biomedical Research, Royal Tropical Institute, Amsterdam, The Netherlands. 4 - Unilever Research and Development, Advanced Measurement and Imaging, P.O. Box 114, 3130 AC Vlaardingen, The Netherlands.

A fast gas chromatography-mass spectrometry (GC-MS) method with minimum sample preparation is described for early diagnosis of tuberculosis (TB). The automated procedure is based on the injection of sputum samples which are then methylated inside the GC-injector using thermally assisted hydrolysis and methylation (THM). The THM-GC-MS procedure was optimized for the injection of sputum samples. For the identification of Mycobacterium tuberculosis the known marker tuberculostearic acid (TBSA) and other potentional markers were evaluated. Hexacosanoic acid in combination with TBSA was found to be specific for the presence of M. tuberculosis. For validation of the method several sputum samples with different viscosities spiked with bacterial cultures were analyzed. The detection limit was better than 1 x 104 bacteria/ml. 17 methyl octadecanoic acid was used as standard. The detection limit for this compound was 20 pg/ml. Finally, 18 stored sputum samples collected in Vietnam from patients suspected to suffer from TB were re-analyzed in Amsterdam by microscopy after decontamination/concentration and using the new THM-GC-MS method. No false positives were found by THM-GC-MS and all patients who were diagnosed with TB were also found positive using our newly developed THM-GC-MS method. These results show that the new fast and sensitive THM-GC-MS method holds great potential for the diagnosis of TB. This work was partially supported by the Foundation of New Diagnostics (FIND) and the Optimus Foundation.

56 ESM 2009 OP-15

HOW WILD-TYPE MIC DISTRIBUTIONS CAN BE USEFUL TO DETERMINE CLINICAL BREAKPOINTS IN Mycobacterium tuberculosis

Ängeby, Kristian 1, Juréen, Pontus 2, Giske, Christian 1, Chryssanthou, Erja 1, Werngren, Jim 2, Hoffner, Sven 2, Kahlmeter, Gunnar 3, Sturegård, Erik 4, Schön, Thomas 5 1 - Department of Clinical Microbiology, Karolinska University Laboratory and Karolinska Institute, Sweden 2 - Department of Bacteriology, Swedish Institute for Infectious Disease Control, Sweden 3 - Department of Clinical Microbiology, Växjö Hospital, Sweden 4 - Department of Clinical Microbiology, Malmö University Hospital, Sweden 5 - Department of Clinical Microbiology, Kalmar County Hospital, Sweden

The increasing prevalence of multidrug resistant (MDR) and extensively drug resistant (XDR) tuberculosis (TB) un- derscores the need for accurate drug susceptibility testing (DST) .It is unfortunate that the current critical antibiotic concentrations (breakpoints) are based mostly on empiry and only to a limited extent on scientific evidence. This is especially true for second line drugs. For most other bacterial pathogens, wild-type minimal inhibitory concentration (MIC) distributions has been successfully applied as one of other tools (such as pharmacokinetic and pharmacodynamic data) to determine clinical breakpoints for DST. A microorganism is defined as wild-type by the absence of acquired and mutational resistance mechanisms to the drug in question. In modern breakpoint determination, breakpoints that divide divide wild type distributions of MICs are avoided. An isolate with a MIC above the wild-type distribution is highly likely to harbor resistance mechanisms and is considered clinically resistant until there is evidence to the contrary. At present, wild-type MIC-distribution data for M. tuberculosis is scarce, presumably in part because of the complexity of DST. In order to establish wild-type MIC distributions for M. tuberculosis we used a 96-stick replicator method, which allowed us to efficiently determine the MICs of 95 clinical isolates simultaneously for four first line drugs (isoniazid, rifampicin, ethambutol and streptomycin) and 15 second line drugs (including four quinolones, four injectables, ethionamide, prothionamide, cykloserine, PAS, thioacetazone). Our findings clearly demonstrate how wild-type MIC distributions among other tools can be used to define clinical breakpoints also in M. tuberculosis.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 57 OP-16

MOLECULAR TECHNIQUES TO MONITOR TB PATIENTS’ TREATMENT: SELECTIVE REMOVAL OF DNA FROM DEAD BACTERIA IN MIXED POPULATIONS BY USE OF ETHIDUM MONOAZIDE

Miotto Paolo, Cirillo Daniela M. Emerging Bacterial Pathogens Unit, San Raffaele Scientific Institute, Milan – ITALY

Drug-resistant tuberculosis (TB) represents a public health problem worldwide and is considered a real threat for TB control programs. Modern nucleic acid amplification techniques (NATs) that exploit nucleic acids signals from clinical samples represent useful tools to allow rapid detection of resistant M. tuberculosis strains. Identification of resistant bacteria in clinical samples is fundamental in order to prevent inadequate treatment and further development of resistant strains. However, NATs can not be used for patients’ follow-up because DNA-derived signals can originate from nonviable bacterial cells and, therefore, generate data that could be misinterpreted. A method developed for microbial food pathogens and already tested on environmental samples is here tested to distinguish between live and dead mycobacteria. Ethidium monoazide bromide (EMA) is membrane impermeant that intercalates into both extracellular DNA and DNA in nonviable cells and is excluded from viable bacteria. Exposure to a light source renders EMA-DNA incapable of con- tributing to PCR. Liquid culture of M. tuberculosis H37Rv was heat inactivated by incubation at 95 °C for 30 min and then treated with 6 μM, 9 μM, 12 μM, respectively, EMA concentrations. EMA treatment consists of 20 min of incubation at +4 °C in the dark followed by 30 min of exposure to a 500 Watt light. Controls used in the study were: inactivated bacteria untreated with EMA, live bacteria untreated with EMA, and live bacteria treated with the same 3 EMA concentrations. After EMA treatment, samples and controls were processed for DNA extraction by thermal lysis (95 °C for 30 min) and directly used in PCR. Amplicons were analyzed by capillary electrophoresis (Agilent Technologies). EMA treatment at all three concentrations tested suppressed PCR amplification from heat inactivated cultures without affecting amplification from live bacteria, proving that EMA could be used also for mycobacterial samples. Preliminary studies were also conducted on mixed live/dead populations using two strains with a different MIRU-VNTR genotype. Performing MIRU typing on mixed samples after EMA treatment, only the live strain could be detected. Comparison of treated and untreated samples highlighted the significant contribution that nonviable bacteria can make to DNA-based diagnostic analysis. EMA approach could be a simple and effective means to monitor patients’ treatment allowing the use of NATs during follow-up.

58 ESM 2009 OP-17

Mycobacterium tuberculosis IS ABLE TO ACCUMULATE AND UTILIZE CHOLESTEROL

Brzostek Anna 1,2, Pawelczyk, Jakub 2, Rumijowska-Galewicz, Anna 2, Dziadek, Bozena 3, Dziadek, Jaroslaw 2 1 - Laboratory of Mycobacterium Genetics and Physiology PAS, Institute for Medical Biology 2 - PAS, Institute for Medical Biology 3 - University of Lodz, Department of Immunoparasitology

Mycobacterium tuberculosis, is the causative agent of tuberculosis that infects one third of the human population. Tubercle bacilli are able to persist in a dormant state, from which they may reactivate disease state. The presence of lipid me- tabolism genes in the genome of M. tuberculosis suggests that lipids, including steroids, are important carbon and energy sources for this pathogen. One potential nutrient that is available in the mammalian host is cholesterol, a major sterol of the plasma membrane. Cholesterol is essential for the uptake of mycobacteria by macrophages, and it has been found to accumulate at the site of M. tuberculosis entry. Moreover cholesterol can be utilized by fast-growing, non-pathogenic mycobacteria, but pathogenic mycobacteria might not be able to use cholesterol. Here, we show for the first time that M. tuberculosis grown in media containing carbon source other than cholesterol is able to accumulate cholesterol in the free lipid zone of its cell wall. This cholesterol accumulation decreases the permeability of the cell wall for the primary anti- tuberculosis drug, rifampin, and partially masks the mycobacterial surface antigens. Furthermore, M. tuberculosis was able to grow on mineral media supplemented with cholesterol as the sole carbon source. Targeted disruption of the Rv3537 (kstD) gene inhibited growth due to inactivation of the cholesterol degradation pathway, as evidenced by accumulation of the intermediate, 9-hydroxy-4-androstene-3,17-dione. Our findings that M. tuberculosis is able to accumulate cholesterol in the presence of alternative nutrients and use it when cholesterol is the sole carbon source in vitro may facilitate future studies into the pathophysiology of this pathogen. The work was supported partially by grants ICGEB (CRP/POL07-01) and State Committee for Scientific Research (N302 035 31/3172).

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 59 OP-18

MYCOLIC ACID-INDUCED IFN-γ PRODUCTION BY CD1-RESTRICTED T CELLS FROM TUBERCULOUS PATIENTS

Rodríguez-Güell, E 1, Alonso, C 2, del Val-Romero, B 2, Clivillé, R 2, Secanella,SP 1, Roura-Mir,C 3, Cañete, C 4, Navarro, A 4, de Gispert, FX 4, Luquin, M 1, Julián,E 1 1. Dept. Genètica i Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra (Barcelona) 2. Dept. Microbiologia, Consorci Sanitari Integral, L’Hospitalet de Llobregat (Barcelona) 3. Dept. Fisiologia i Immunologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra (Barcelona) 4. Unitat de Respiratori, Consorci Sanitari Integral, L’Hospitalet de Llobregat (Barcelona)

Introduction The cell wall of Mycobacterium tuberculosis (MTB) has a large number of structurally diverse lipids. Mycolic acids (MAs) merit special interest because their immunogenicity has been demonstrated in CD1-restricted T cell clones; specifically, they are presented by CD1b molecules. To date, the recognition of MAs in tuberculous patients (TB) has not been studied. The purpose of the study was to determine whether MAs are recognized by the immune system of TB patients and, in the event of this being so, to study the evolution of this response throughout anti-TB treatment.

Methods Immature dendritic cells (iDCs) isolated from 31 TB patients at the time of diagnosis and throughout anti-TB treatment, 20 PPD-positive and 20 PPD-negative donors were analysed by cytometry for CD1b surface expression. Subsequently, the samples were irradiated and cultured with autologous lymphocytes in the presence of phytohemaglutinin, MTB and purified MAs as stimuli. After 48 hours, IL-10 and IFN-γ were quantified by enzyme-linked immunosorbent assay.

Results No significant differences among the surface expression of CD1b in iDCs from healthy donors or TB patients were observed at any time during the disease. iDCs from all the individuals were therefore able to present MAs. Median levels of stimuli-induced IL-10 in samples obtained at the outset of anti-TB treatment were lower than those obtained at the end; however, no significant differences were observed. Nor were any differences observed among the IL-10 levels obtained from the different groups of individuals. In TB patients, MA-induced IFN-γ median values obtained at the end of prophylaxis were significantly higher, statistically, than those elicited at the beginning. Grouping the samples of the 31 TB patients in terms of collection time, IFN-γ median levels followed an upward trend throughout anti-TB treatment, reaching maximum levels at the point of disease cure. Furthermore, in PPD-negative donors IFN-γ median values were lower than those from PPD-positive donors, significant differences only being established when MTB was used as antigen.

Conclusions The specific cellular immune response against MAs in TB patients described here for the first time suggests a potential immunoprotective role for MAs.

60 ESM 2009 OP-19

A REPORT ON NEW ADAPTED FORMS OF EXTENSIVELY DRUG RESISTANCE TUBERCLE BACILLI : TRANSMISSION ELECTRON MICROSCOPY ANALYSIS

Parissa Farnia(PhD)1. Ali Akbar Veleyati (MD)1, Mohammal Reza Masjedi (MD)1, 2 Tengku Azmi Ibrahim (PhD)2, Payam Tabarsei (MD), Rafiuz Zaman Haroun (MSC)2, Ho Oi Kuan (MSC))2, Abdul Rahman Omar (PhD)1 1 - Mycobacteriology Research Centre, National Research Institute of Tuberculosis and Lung Disease (NRITLD), WHO Collaborating Centre, Shahid Beheshti University (Medical Campus), Darabad, Tehran ,19556, P.O: 19575/154, Iran. E-mail: [email protected] 2 - Microscopic unit, Institute of Bioscience , University Putra Malaysia ,43400 UPM ,Serdang, Selangor Darul Ehsan , Malaysia

Background Extensively drug resistance tuberculosis bacilli (XDR-TB), is caused by a strain of M. tuberculosis(MTB) that are resistant to isoniazid and rifampin (which defines MDR tuberculosis) in addition to any fluroquinolone and at least one of the three following injectable drugs: caperomycin, kanamycin and amikacin. Viewed under transmission electron microscopy (TEM), spore like structure was observed inside the XDR-TB bacilli.

Methods The susceptibility testing against first and second line drugs was performed on isolated M. tuberculosis strains. Subsequently, a homogenous thick suspension of 107 to 108 cells at exponential phase was prepared and observed under Transmission Electron Microscopy (TEM). Five isolates from each group (susceptible, MDR, and XDR TB) were used in this study.

Results Viewed under the TEM, the XDRTB bacilli at exponential phase had three types of cell division 1) 80-70% of bacilli were looks as norm one with symmetrical or asymmetrical cell division 2) 5-7% with extra ordinary thick cell wall ( 21 to 26 nm ) which was similar to stationary or dormant phase bacilli. But surprisingly the stationary phase XDR-TB bacilli were at dividing process 3) 15-20% bacilli had spore formation inside them These spores were different from buds or a polar division that formed during cell branching of MTB. No spore’s and stationary phase bacilli were detected in susceptible or multidrug resistant strains of MTB.

Discussion Dividing phenomena in XDR-TB bacilli with stationary phase appearance will generate new adapted types of M. tuberculosis which can resist the effect of all available anti tuberculosis drugs. At the same time, it is possible that under certain conditions, the XDR-TB bacilli produce spore to overcome the hostile environment. Spore formation in XDR-TB bacilli should take very seriously from epidemiological point of view. With these new forms of adaptation that occurs in XDRTB bacilli , how should we treat and control the diseases.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 61 OP-20

GROWTH PROFILE OF CLINICAL ISOLATES OF Mycobacterium tuberculosis COMPLEX IN MURINE MACROPGHAGES

Susanne Homolka1, Stefan Niemann1, David G. Russell2 and Kyle H. Rohde2 1 - Molecular Mycobacteriology, National Reference Center of Mycobacteria, Borstel Germany 2 - Department of Microbiology/Immunology, Cornell University, Vet. Med. Center, Ithaca, USA

Background Mycobacterium tuberculosis and other members of the Mycobacterium tuberculosis complex (MTC) remain a major cause of morbidity and mortality worldwide. Several studies based on spoligotyping, IS6110 fingerprinting and MIRU-VNTR typing demonstrated that the global population structure of MTC is defined by phylogeographical lineages and genotypes that are also associated with pathogenic differences. However, the influence of strain genomic variation on the outcome of infection and the clinical presentation are not completely understood. In our study, we investigated growth differences of 15 strains of five different genotypes in comparison to the CDC1551 reference strains in liquid culture and in macrophages.

Methods Murine bone marrow derived macrophages were infected with liquid culture of different clinical isolates (MOI 3:1; 2x106CFU/ml). Survival of strains in resting and activated macrophages was determined at different time points up to 11 days post-infection. Additionally, growth kinetics of all strains in 7H9 liquid media were analyzed.

Results Growth analyses of clinical isolates in liquid culture based on OD measurements showed no significant differences in comparison to CDC1551. However, we observed both strain- and genotype-specific survival and growth profiles within resting and activated macrophages.

Conclusions The genomic diversity of clinical isolates influences the complex interaction of the pathogen with host phagocytes. Further analyses to correlate intracellular gene expression profiles with the outcome of infection are in progress.

62 ESM 2009 OP-21

PRESENCE OF ESAT-6 AND CFP-10 GENES DOES NOT LEAD TO PHAGOLYSOSOME TRANSLOCATION OF Mycobacterium szulgai

Jakko van Ingen1,2, Nicole van der Wel3, Richard Dekhuijzen2, Martin Boeree2, Dick van Soolingen1 1 - National Mycobacteria Reference Laboratory, National Institute for Public Health and the Environment, Bilthoven, the Netherlands 2 - Department of Pulmonary diseases, Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands 3 - Department of Cell Biology, Netherlands Cancer Institute, Amsterdam, the Netherlands

Background Bacterial virulence factors in nontuberculous mycobacteria are mostly unknown. In Mycobacterium tuberculosis complex bacteria, the esat-6 and cfp-10 genes are important virulence factors, which facilitate translocation from the phagolysosome to the cytosol of macrophages. Their presence and role among nontuberculous mycobacteria is largely unknown.

Methods We assessed the presence of esat-6 and cfp-10 genes in all 5 M. kansasii subtypes based on 16S-23S internal transcribed spacer sequencing (n=15), M. szulgai (4), M. marinum (4), M. avium (2), M. conspicuum (4), M. genavense (1), M. bohemicum (2), M. interjectum (2), M. flavescens (5), M. xenopi (2), M. malmoense (2), “M. riyadhense” (1) and M. tuberculosis H37Rv by PCR. We used Esa-12 CATGACAGAGCAGCAGTG and Esa-303 5’-GCCCTATGCGAACATCCC-3’ primers for esat-6 and opBR78 5’-GTAGCCCGGGATGGCAGAGATGAAGACCGATGCC-3’ and opBR103 5’-TCAGAAGCCCATTT- GCGAGGACAGC-3’ primers for cfp-10. For PCR negatives, we performed Southern blotting with probes based on the esat-6 gene of M. tuberculosis H37Rv. One NTM species with esat-6 and cfp-10 genes was selected for macrophage infection. By cryo-immunogold electron microscopy we tested whether these genes effect translocation from the phagolysosome to the cytosol of macrophages.

Results We were able to amplify esat-6 and cfp-10 genes in M. tuberculosis H37Rv, all M. kansasii subtypes, M. szulgai, M. marinum and “M. riyadhense”. All esat-6 and cfp-10 sequences among these nontuberculous mycobacteria were species-specific; multisequence alignment revealed an average of 90% homology with the M. tuberculosis sequences. The remaining species were repeatedly negative by both PCR and Soutern blotting. Mycobacterium szulgai was subsequently used for a macrophage (THP-1) infection experiment, with an M. tuberculosis positive control. Seventy-two hours after infection, no cytosolic M. szulgai bacteria were found, while 53% of the M. tuberculosis bacteria had translocated to the cytosol.

Conclusions While some nontuberculous mycobacteria harbor esat-6 and cfp-10 genes, their products, at least for M. szulgai, do not effect translocation of the bacteria from the phagolysosome to the cytosol in macrophages. ESAT-6 and CFP-10 protein structure or secretion could be critical factors. While the presence of esat-6 and cfp-10 genes adds an interesting phylogenetic marker, the pathogenesis of NTM infections remains unexplained.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 63 OP-22

DEVELOPMENT OF AN ADAPTIVE IMMUNE RESPONSE IN THE DRAINNG LYMPH NODE DURING Mycobacterium ulcerans INFECTION

Alexandra G. Fraga; Joana E. Braga; Andrea Cruz; Teresa G. Martins; Daniela R. Pereira; Wayne M. Meyers; Françoise Portaels; António G. Castro; Jorge Pedrosa 1 - Life and Health Sciences Research Institute (ICVS). School of Health Sciences, University of Minho. Braga, Portugal 2 - Mycobacteriology Unit, Department of Microbiology, Institute of Tropical Medicine, Antwerp, Belgium 3 - Armed Forces Institute of Pathology, Washington, D. C., USA

Buruli ulcer is a neglected tropical disease caused by infection with Mycobacterium ulcerans. The necrotic cutaneous lesions of BU patients are associated with the cytotoxic properties of the exotoxin mycolactone. Previous results from our group show that the protective role of IFN-γ is impaired during infection with the highly virulent strain 98-912. To further characterize the development of an adaptive immune response during progressive infection with a highly virulent M. ulcerans strain, we decided to study the T cell dynamics in the draining lymph node (DLN) of mice infected with M. ulcerans 98-912, as compared to the low virulent, mycolactone-deficient strain 5114. Subcutaneous infection of mouse footpads with M. ulcerans 5114 induced a modest increase in the number of cells in the DLN, prevalent throughout the experimental infection. Surprisingly, infection with strain 98-912 led to a 26-fold increase in the number of cells in the DLN, followed by a significant drop to basal levels. However, this increase in the number of cells did not correlate with a protective response in the infected footpad. Following this observation, we explored whether the induction of an adaptive immune response occurs during infection with strain 98-912. M. ulcerans infection elicited antigen-specific T cells producing IFN-γ in the DLN, with the response being more prominent in M. ulcerans 98-912 infected mice. Additionally, infection with M. ulcerans followed by adoptive transfer of OVA-transgenic T cells did not impair the accumulation, proliferation or activation of the OVA-specific T cells in the DLN upon challenge with OVA. Moreover, this T cell accumulation and activation was significantly increased in mice infected with strain 98-912. Interestingly, analysis of the DLN at later time points revealed the presence of viable bacilli that correlated with the dramatic decrease in the number of cells. This decrease was due to apoptosis, as demonstrated by the higher number of activated-caspase 3 positive cells. Supporting our observations in the mouse model, presence of bacilli and tissue destruction were also observed in DLN of BU patients. In summary, the failure of the host to generate a protective response against infection with this highly virulent strain of M. ulcerans is not associated with an impaired development of specific T cells in the DLN, but instead to their destruction in the infection foci and, later, to the destruction of the DLN itself.

64 ESM 2009 OP-23

TUBERCULOSIS SOFTWARE IN A GENERAL HOSPITAL, WORKING INSTRUMENT

Portugal Clara 1; Nuno Cardoso 2, Luísa Sancho 1; Germano Sousa 1 1 - Laboratory of Microbiology, Department of Clinical Pathology 2 - Planning and Management Control Director Hospital Fernando Fonseca (HFF) - Amadora, Portugal [email protected]

Background Our Hospital is located in a Lisbon’s surroundings, its construction was completed in 1995 and, in this same year, it started the operation. The HFF was designed to house a population much smaller than that now covers, 750 000. Our population has a low socioeconomic conditions and a high level of immigrants from Africa and in Eastern Europe, which may be the main reason for concentrating a large number of tuberculosis’s cases. Portuguese Health Authorities reported that 66% of TB cases in Portugal were concentrated in Lisbon’s surroundings. In a few years after the opening of the Hospital, all laboratory’s data relating to tuberculosis became too numerous to be recorded and cross in the general program.

Purpose Given the need to gather and cross all the tuberculosis’s data, in 2000 was developed a software (MYCOHFF) that we are going to present.

Methods MYCOHFF results from the interconnection of 3 Excel files; MYCOYEAR related to the year in question, MYCOZIEHL that brings only patient’s data with positive Ziehl-Nielseen (since 1997) and MYCOCULT that joins only the patient’s data with positive cultural examinations (liquid and/or solid) (since 2000). For each specimen submitted for mycobacterial culture, we record the name of the patient, process number, analysis number, product’s type, the origin patient’s Service and product inoculation’s date. Thus takes place an observation’s weekly list. For each week, we note the medium (liquid and Lowenstein-Jensen) growth or not, the contamination and/ or degradation, during 6 or 8 weeks (sometimes more). The year’s records are placed in alphabetical order (patient’s name). The patient’s analysis with positive culture (liquid and/or solid) automatically appears in red. If the patient had already a positive culture, all the negative analysis appears in purple. If the patient had a positive Ziehl- Nielseen, then all the analysis will appear in yellow, becomes purple if it has other positive culture or red if this analysis is positive. On patient’s first isolation, we perform the Identification and Susceptibility Test. This date is noted as well as the final date with the definitive results, which are presented to the clinician.

Results • Warning Signs: When the patient’s registration happens, we know immediately, with different colors (program alerts), if the patient has Ziehl-Nielseen positive, culture positive, and when. • Search: Any search is fast because we are talking about an application that is made in Excel software. • Weekly observation’s list: The weekly sheets are easily created by filtering the inoculation’s date field. • Statistical indicators and graphics: With only a few records, statistical indicators are automatically calculated and graphical analyses are available at any time.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 65 Statistical indicators for each year are concerning to different parameters. 1- Concerning the analysis/patient relationship: Nº of patients, nº of analysis, total positive patients, total positive analysis, analysis/patient ratio, patients positive rate, analysis positive rate; 2- Concerning the Ziehl-Nielseen (Z) and liquid/solid medium relationship: Nº of positive direct tests (Z+), Nº of positive cultural tests (C+), true Z positive’s (Z+/C+) rate, false Z negative’s (Z-/C+) rate, false Z positive’s (Z+/C-) rate, real Z negative’s (Z-/C-) rate, Ziehl-Nielseen’s positivity rate per analysis and per patient; 3- Concerning the identification and TSA: Nº of Mycobacterium sp. isolated, total identified strains, total strains with the TSA and identification still in study, Nº and rate of Mycobacterium tuberculosis complex (CMT), CMT without resistance, with one resistance, MDR (Multi Drug Resistant - TB), XDR (Extensively Drug Resistant – TB), Nontuberculous mycobacteria, average response time beginning with the isolation culture (identification plus TSA). The statistics graphs show: 1 - Analysis requested / positive analysis by organic product; pleural biopsy, pleural fluid, sputum, bronchial secretions, bronchoalveolar lavage, gastric fluid (respiratory specimens), urine, pus, mieloculture, cerebrospinal fluid, biopsy , ascites fluid, fluids, blood; and their positivity, 2 - The isolated Mycobacterium sp.’s strains, including the differentiation in CMT, zero resistance, one resistance, MDR, XDR, 3 - Compare the tuberculosis’s incidence rate in two populations, MDR and non-MDR, for the age range of patients, sex and HIV.

Conclusions For us, MYCOHFF software is an essential tool for the process of gathering and managing tuberculosis data. Quick reports of any patient are possible. In a few seconds, we can make the patient laboratory story on tuberculosis. Each year a statistical report is communicated to all our hospital’s departments. Monthly, or when requested, for the CDP (centre that follows the patients in the community), is sent a list identifying new positive cases and results (preliminary and/or final).

66 ESM 2009 OP-24

Development of an automated culture system for M. tuberculosis with autofluorescence detection den Hertog, Alice 1, Koeleman, Marc 1, Ingham, Colin 2, Fey, Frank 3, Langerak, Edwin 3, Klatser, Paul 1, Anthony, Richard 1 KIT Biomedical Research, Amsterdam, The Netherlands Microdish BV, Wageningen, The Netherlands CCM, Nuenen, The Netherlands

Due to the unavailability of rapid sensitive diagnostic methods for tuberculosis (TB), culture remains one of the most important diagnostic tools - even though it is technically demanding and slow. The time required for culture leads to a delay in TB diagnosis and treatment with effective drugs. A standardized method for more rapid drug sensitivity testing (DST) of TB would be valuable. We are developing a system in which mycobacterial (micro-)colonies are detected by their autofluorescence based on the MODS (microscopic observation of drug susceptibility) method, which is much more rapid than the traditional culture methods, but is laborious. By using microscopy with low-power magnification, colony growth could be determined much earlier than when viewed by eye. Studying Mycobacterium smegmatis initially as a model organism, we have made se- quential brightfield and fluorescence microscopy photographs of colonies growing on movable solid supports on media. These images were used to develop an image analysis protocol to determine the growth rate. We found that the time to detection of microcolonies was less than half that required for visual detection. Furthermore, as soon as the colonies could be visualized, further growth of colonies could be detected by the image analysis protocol within 1-2 division times, from which the growth rate could be determined. As the bacteria were inoculated on movable solid supports, exposing the microcolonies to selective media after their first detection could make DST of many individual colonies possible after only a few additional division times, allowing the rapid determination of the proportion of antibiotic resistant and sensitive mycobacterial colonies present in a sample. After confirmation of the recently described autofluorescence of mycobacteria, this method was used for the detection and enumeration of viable mycobacteria. The specificity of the autofluorescence may be used to confirm the presence of mycobacteria. The use of (semispecific-) autofluorescence in combination with growth rate will increase the feasibility of developing an automated detection system based on this method, which is very challenging for light microscopy methods (such as MODS). Additionally, the possibility of performing quick DST without re-inoculation may lead to a reduce time to correct treatment.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 67 OP-25

SECOND-LINE DRUG SUSCEPTIBILITY TESTING OF Mycobacterium tuberculosis BY MGIT 960 SYSTEM, THE MICROPLATE COLORIMETRIC-BASED METHOD AND THE PROPORTION METHOD

Nora Morcillo1, Belén Imperiale,1, 2, Beatriz Di Giulio3 1 - Reference Laboratory of Tuberculosis Control Program of Buenos Aires Province, Dr. Cetrángolo Hospital Buenos Aires, Argentina. 2 - National Council of Scientific and Technological Research, Buenos Aires City. 3 - P. V. de Cordero Hospital, San Fernando, Buenos Aires, Argentina.

The accurate treatment of tuberculosis (TB) cases due to multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis emphasizes the necessity of new tools for rapid detection of these strains in clinical laboratories. Minimal inhibitory concentrations (MICs) by MGIT960 and the colorimetric microplate method using dyes as MTT or resarzurin (CMM) were determined for the following drugs (µg/ml): amikacin (AMK): 2.0, 4.0, 8.0; kanamycin (KM), capreomycin (CPM), ethionamide (ETH): 2.5, 5.0, 10.0; cycloserine (CS): 15.0; ofloxacin (OFX) and linezolide (LZ): 0.5, 1.0, 2.0; and moxifloxacin (MOX) 0.25, 0.5, 1.0. MICs were performed on 94 clinical isolates. The proportion method on Middlebrook 7H11 (PM) was used as gold standard. Inoculated MGITs were incubated in the instrument for no longer than 21 days. A strain tested by MGIT960 was considered resistant if a positive signal flagged from the drug-containing tube within 5 days of the positive control tube. Microplates of the CMM were incubated for an average of 8 days. Statistical methods were applied to define drug-resistant strains on the basis of the comparison between results obtained by MGIT960 and CMM with the PM. The following critical concentrations were identified (µg/ml): AMK: 4.0; CPM, ETH and KM: 5.0; CS: 30.0; LZ: 1.0; MOX: 0.5; OFX: 2.0. Accuracy of MGIT960 and M-MTT was 100% for AMK, CPM, OFX, MOX and LZ. In this study tubes incubation and positivity detection was manually obtained from the MGIT960 instrument which actually can be adapted to automatically detect both susceptible and resistant strains to each one of the second-line drugs. Results were obtained in less than 10 days for both MGIT960 and CMM. On the other hand CMM, as a complete homemade method, was cheaper but more laborious than MGIT960. Our results showed that both methods could be promissory implemented as a rapid diagnosis tools to detect MDR and XDR-TB cases in clinical practice.

68 ESM 2009 OP-26

THIORIDAZINE SHOWS PROMISING ACTIVITY IN A MURINE MODEL OF MULTIDRUG-RESISTANT TUBERCULOSIS

Jakko van Ingen1,2, Martin Boeree1, Leonard Amaral3, Rogelio Hernandez Pando4, Dick van Soolingen2 1 - Department of Pulmonary Diseases, Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands 2 - National Mycobacteria Reference Laboratory, National Institute for Public Health and the Environment, Bilthoven, the Netherlands 3 - Mycobacteriology Unit, Institute of Hygiene and Tropical Medicine, Universidade Nova de Lisboa, Lisbon, Portugal 4 - Experimental Pathology Section, Department of Pathology, National Institute of Medical Sciences and Nutrition Salvador Zubiràn, Mexico City, Mexico

Background Multidrug-resistant tuberculosis (MDR-TB) is a threat to TB control efforts worldwide for which very few active drugs are currently available. The phenothiazines are antipsychotic agents that have potential as anti-tuberculosis drugs, at least in vitro. Within this pharmacological class thioridazine is the most efficacious and causes the mildest side-effects when used as an antipsychotic agent. Thioridazine is active against mycobacteria by targeting the type II NADH dehydrogenase, succinate dehydrogenase, the binding of calcium to proteins and disruption of aerobic respiration under micro-aerobic conditions. We tested its in vivo activity in a murine model.

Methods We infected 3 groups of 40 Balb/c mice with pansusceptibe M. tuberculosis H37Rv. After 60 days, 20 mice in each group started 2 months of thioridazine monotherapy at 16, 32 and 70 mg/kg dosages; the remaining 20 served as controls. This experiment was repeated using a clinical multidrug-resistant M. tuberculosis (MDR-TB) isolate and two months daily oral administration of 32 and 70 mg/kg. In a third experiment, 3 groups of 20 mice were infected with M. tuberculosis H37Rv; one group received rifampicin, isoniazid and pyrazinamide, another received these 3 drugs and thioridazine, one untreated group served as controls. In all experiments, groups of five mice per group were sacrificed after 2, 4 and 8 weeks of treatment; lung tissue was homogenized for quantitative cultures. The bacillary load of the lungs was determined by colony forming units (CFU) quantification; histological damage was observed by microscopic examination.

Results In both the M. tuberculosis H37Rv and MDR-TB infection, monotherapy with 32 and 70mg/kg thioridazine lead to signifi -H37Rv and MDR-TB infection, monotherapy with 32 and 70mg/kg thioridazine lead to significant reductions in CFU counts from the lung tissue homogenates and in the extent of histological damage, at all time points. ��Moreover, when 32 mg/kg of thioridazine was added to a regimen containing rifampicin, isoniazid and pyrazinamide for susceptible tuberculosis, a significant synergistic effect was achieved.

Conclusions Thioridazine shows promising activity in our murine model. It has a potential effect in the treatment of susceptible and MDR-TB, where few active drugs are available. The low price of this out of patent drug enables extended use in resource–poor settings where the burden of multidrug-resistance is most grave.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 69 OP-27

CONTRIBUTION OF EFFLUX PUMP ACTIVITY FOR MACROLIDE RESISTANCE IN M. avium COMPLEX

Liliana Rodrigues1,2*, Daniela Sampaio1, Isabel Couto1,3, Diana Machado1, Winfried V. Kern4,5, Leonard Amaral1,2,5 and Miguel Viveiros1,5 1 - Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Lisbon, Portugal 2 - UPMM, IHMT/UNL, Lisbon, Portugal 3 - Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e Tecnologia, UNL, Caparica, Portugal 4 - Center for Infectious Diseases and Travel Medicine, University Hospital, Freiburg, Germany 5 - COST ACTION BM0701 (ATENS)

Mycobacterium avium complex (MAC), comprising M. avium and M. intracellulare, is clinically important since it can cause severe infections in AIDS patients and other immunocompromised individuals. Therapy of MAC infections is problematic due to the intrinsic resistance of these bacteria to many of the available antimicrobial drugs. The use of the macrolides clarithromycin and azithromycin has improved the outcome of MAC infections, but therapeutic failure is still a major problem. We have recently shown that efflux pumps of MAC play an important role on this resistance phenotype. In fact, increased activity of efflux pumps is known to contribute to a multidrug resistance phenotype by extruding a wide variety of chemically and structurally unrelated compounds from the cell, preventing them from reaching their cellular targets. Thus, the characterization of such efflux pumps is crucial for the design of new antimycobacterial therapeutic strategies. In this work, we have studied the efflux pump activity in MAC clinical strains by a fluorometric method that detects efflux activity on a real-time basis, and evaluated the contribution of active efflux to the resistance to macrolides. The results to be presented show that resistance to clarithromycin was significantly reduced in the presence of efflux pump inhibitors (EPIs) such as the calcium-channel inhibitors thioridazine or chlorpromazine and the calcium ion influx inhibitor verapamil. The same EPIs were effective in decreasing the efflux of ethidium bromide (a common efflux pump substrate) from MAC cells, as shown by fluorometric analysis. Moreover, the retention of [14C]-Erythromycin by the same inhibitors demonstrated that active efflux contributes to MAC resistance to macrolides. In conclusion, this study demonstrates that efflux pumps play an important role in MAC resistance to antibiotics, particularly to macrolides, and opens the possibility to explore the usefulness of these EPIs, already used in clinical practice for other purposes, such as thioridazine, as adjuvants to enhance the effectiveness of the therapeutic regimens against MAC infections.

70 ESM 2009 OP-28

MEMBRANE- BASED VERSUS MICROBEAD- BASED SPOLIGOTYPING: PRELIMINARY RESULTS ON A QUALITY- INSURANCE STUDY ON 10 SITES WORLWIDE

Abadia E1, Zhang J1, Refregier G1, Sola C1. 1 - Institut de Génétique et Microbiologie, UMR8621, CNRS-Université Paris-Sud (Universud), Equipe IGEPE, bât. 400. France.

Spoligotyping have been developed 12 years ago and have been used worlwide for molecular epidemiological or molecular evolutionary studies. This technique is based on a reverse line- blot hibridization method. Due to it’s wide accep- tance (458 references in Pubmed on 2009 April 27th) it can be considered as a basic technique in genotyping strains of Mycobacterium tuberculosis complex together with MLVA (Multi locus variable analysis) typing. However, spoligotyping has not been the focus of standarization nor of quality insurance studies. The transfer from the membrane- based to a microbead- based format, through the development of the multiplex Luminex plataform was done in 2004 in the CDC in Atlanta. We implemented this technique in our team in 2008. We wanted to evaluate membrane- based spoligotyping results obtained on 10 sites (around 1000 DNA samples from clinical isolates) comparing them with those from the Luminex microbead- based results obtained on the same samples. The DNA samples were selected to fulfill the following goals: (1) Retrospectively assess the quality of spoligotyping results, that have been produced in various laboratories worlwide during a decade (2) Solve inconsistencies, resolve discrepancies, improve quality of hard- to- interprete membrane- based spoligotyping results (3) Study if DNA extraction procedure (quality, quantity) may have produced errors in spoligotyping patterns production (4) assess the economical interest of the new Luminex- based technique and it’s contribution (or not) to and increased quality of services in molecular epidemiological studies. We will present all results available until today. Our current experience and preliminary results suggest that the Luminex platform it’s very versatile to produce high quality spoligotypnig results with both, a higher throughput and sensitivity than the membrane- based spoligotyping and at affordable costs for developed countries.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 71

Abstracts of POSTER PRESENTATIONS (PP)

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 73

PP-1

EVALUATION OF THE HIGH-THROUGHPUT REPETITIVE-SEQUENCE-BASED PCR DIVERSILAB SYSTEM IN M. tuberculosis MOLECULAR EPIDEMIOLOGY STUDIES

Miotto Paolo, Baldan Rossella, Cirillo Daniela M. Emerging Bacterial Pathogens Unit, San Raffaele Scientific Institute, Milan – ITALY

PCR-based methods have been developed to simplify and reduce the time required for genotyping M. tuberculosis by standard approaches based on IS6110-Restriction Fragment Length Polymorphism (RFLP). Of these, MIRU-VNTR complemented with spoligotyping has been proposed as an alternative. Repetitive-sequence-based PCR (rep-PCR) is useful for generating DNA fingerprints of diverse bacterial species. Rep-PCR amplicon fingerprints represent genomic segments lying between non-coding repetitive sequences. A commercial system (Diversilab, Biomerieux) that electrophoretically separates rep-PCR amplicons on microfluidic chips, and provides computer-generated readouts of results has been adapted for use with Mycobacterium species. The ability of this system to type M. tuberculosis was evaluated in comparison with spoligotyping and MIRU-VNTR in two different panels. First, we evaluated a 35 strains panel by MIRU-15 complemented with spoligotyping and Diversilab rep-PCR. Results were analyzed with MIRU-VNTRplus database for spoligo-MIRU, and with Diversilab Software for rep-PCR using two different algorithms (Pearson Correlation [PC] and Kullback-Leibler [KL]). Threshold for clusters was fixed at 98% of similarity for rep-PCR. MIRU-15 showed a clustering rate of 11.4% whereas the rep-PCR reported a clustering rate of 28.6% (PC) and 17.1% (KL). The discriminatory power (Hunter-Gaston discriminatory index [HGDI]) for spoligo-MIRU- 15 resulted 0.983 whereas for rep-PCR was 0.978 (PC) and 0.983 (KL). We also compared the two techniques on a panel composed by 8 closely related strains from a probable outbreak. In this case the rep-PCR showed a discriminatory power of 0.571 (PC) and 0.679 (KL), compared to a HGDI of 0.643 for spoligo-MIRU-15. Nevertheless, clustering rate for MIRU-15 was 50.0% whereas rep-PCR algorithms showed a clustering rate of 100.0% (PC) and 62.5% (KL), respectively. To understand the meaning of the discrepancies still found between spoligo-MIRU-15 and rep-PCR, analysis of epidemiological data for the clustered patients will be taken in consideration. Preliminary data obtained by Diversilab suggest that the KL algorithm is more appropriate for M. tuberculosis typing analysis. Nevertheless, even if rep-PCR results analyzed by KL algorithm showed a discriminatory power similar to MIRU-15, clustering rate remains higher. This new technique could be useful for a routine use in clinical laboratories for real-time genotyping and laboratory contaminations control.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 75 PP-2

68 SPACERS Mycobacterium tuberculosis COMPLEX SPOLIGOTYPING : A STUDY USING A MICROBEAD-BASED HIGH THROUGHPUT FORMAT.

Zhang J1, Abadia E1, Refrégier G1 , Ruimy R2, Boschiroli ML3, Guillard B4 and C. Sola1. 1 - Institut de Génétique et Microbiologie, UMR8621, CNRS-Université Paris-Sud (Universud), Equipe IGEPE, bât. 400, Centre scientifique d’Orsay, rue Gregor Mendel, 91405 Orsay-Cedex2 - APHP, Hôpital Bichat-Claude Bernard, Paris 3 - Agence française de sécurité sanitaire des aliments AFSSA, Maisons-Alfort 4 - Institut Pasteur du Cambodge

New captures probes for the microbead-based spoligotyping assay (Luminex) were designed to test for the presence/ absence of 25 spacers that are not used in routine spoligotyping. These spacers are expected to provide an improved discriminatory power for Major Genetic Group I, including the « ancestral » TbD1+ MTC (Mycobacterium tuberculosis complex) clinical isolates. The new 68 spacers spoligotyping format developed on a Luminex platform was shown to give excellent results. Around 400 strains of MTC from 3 different centers (Bichat Hospital, AFSSA, Institut Pasteur of Cambodia) were studied. We show that the 68 spacers format is more discriminant to study the strains of the East African Indian family (EAI) : among 86 strains of the EAI family, it could distinguish 44 clusters compared to 27 clusters by routine 43 spacers spoligotyping. Whereas for a total of 210 clinical isolates of Mycobacterium bovis, we reported 31 types instead of 25, and for a total of 30 “Beijing” clinical isolates, 4 clusters instead of 3 clusters were found. For Mycobacterium africanum, a total of 17 strains were assayed and 11 instead of 9 clusters were found. High-thr(East-African Indian - Indo-Oceanic clade) and other Major Genetic Group I still-undefined spoligotyping signatures. This observation often concerns the first spacer of a string of several missing spacers (border domains). These observations are currently under further investigation by sequencing. Extended High-throughput spoligotyping is also a new mean to detect mutational events that could be diagnostics of clade-specific MTC evolution.

76 ESM 2009 PP-3

ASSOCIATION BETWEEN BEIJING GENOTYPE AND DRUG RESISTANCE AMONG Mycobacterium tuberculosis ISOLATES CIRCULATING IN THE REPUBLIC OF GEORGIA

Stefan Niemann1, G. Khechinashvili2, M. Gegia2, N. Mdivani2, and Y. W. Tang3 Molecular Mycobacteriology, National Reference Center for Mycobacteria, Research Center Borstel, Borstel, Germany Georgian Foundation against Tuberculosis and Lung Diseases, Tbilisi, Georgia Vanderbilt University Medical Center, Nashville, TN, USA

Rising tuberculosis (TB) rates and high levels of multidrug-resistant TB (MDR-TB) have become a major public health problem in several parts of the former Soviet Union. High rates and transmission of MDR-TB have been noticed to be associated with the presence of Mycobacterium tuberculosis Beijing genotype strains pointing to the importance of pathogen genetic factors for the modulation of infection outcome and epidemiology. Here we present the first data on the population structure of M. tuberculosis strains from the Republic of Georgia, a high incidence setting at the Black Sea Coast. All strains were analysed by spoligotyping and 24-loci MIRU-VNTR genotyping. Identification of major M. tuberculosis genotypes was carried out by using the MIRU-VNTRPlus database (www.miru-vntrplus.org) and a similarity analysis performed to identify strains with identical genotyping profiles (clusters), which are indicative for the rate of recent transmission. Anti-tuberculosis drug resistance was determined by in vitro antimicrobial susceptibility testing. Genotyping profiles were successfully generated for 187 M. tuberculosis isolates which were further investigated. The most prominent genotype found, was Beijing (29%), followed by LAM (18%), Ural (12%), and Haarlem (n=10) strains. Approx 50% of the isolates were grouped in clusters. When the distribution of drug resistance is considered, it was noticed that MDR-TB was nearly completely restricted to Beijing strains. Further detailed analyses are currently in progress. Our data underline the importance of Beijing genotype strains for the TB epidemiology in former Soviet Union countries. However, Ural, Haarlem genotype and a large variety of strains of so far undefined lineages represent nearly two third of the strains found in Georgia. Although Beijing strains are not as dominant as in others Eastern European countries such as Kazakhstan, we confirm a clear association between MDR-TB and the Beijing genotype in Republic of Georgia.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 77 PP-4

Mycobacterium tuberculosis EPIDEMIOLOGY AND GENETIC DIVERSITY IN THE TWIN ISLAND REPUBLIC OF TRINIDAD AND TOBAGO

Shirematee Baboolal, 1, 2 Julie Millet,3 Patrick Eberechi Akpaka, 1 Dottin Ramoutar, 4 Nalin Rastogi 3 1 - Department of Para-Clinical Sciences, Faculty of Medical Sciences, The University of the West Indies, St. Augustine, Trinidad & Tobago 2 - Caribbean Epidemiology Centre, Jamaica Boulevard, Port of Spain, Trinidad & Tobago 3 - Unité de la Tuberculose et des Mycobactéries, Institut Pasteur de Guadeloupe, Abymes, Guadeloupe 4 - Caura Chest Hospital, Caura, Trinidad & Tobago

This work describes the first application of molecular tools for studying tuberculosis (TB) epidemiology, genetic diversity, and transmission in the twin island Republic of Trinidad and Tobago (T&T). The study population (n=132) represented one year recruitment of all culture positive TB cases from T&T, and was characterized by a high male to female sex-ratio of 4 (mean age 42.8 years, range 17 to 78 years), and a HIV/TB coinfection rate of nearly 30%. It mainly occurred among African descendants who represented 37.5% of total population but 69.7% of all TB cases (p<0.001). Spoligotyping resulted in 25 different patterns and 12 clusters (2 to 74 strains per cluster). In total, 81.3% of the isolates in our study was defined as modern tubercle bacilli belonging to the Principal Genetic Groups 2/3. Five major lineages were observed: East-African Indian (EAI), Latin-American and Mediterranean (LAM), X, Beijing, and Haarlem. A comparison with international SITVIT2 database showed that the overall lineage distribution in T&T was completely different from other Caribbean neighbors (n=2653 isolates). A high clustering rate of 90% was observed essentially due to a single large cluster of 74 strains designated as Spoligotype International Type - SIT566 (T&T clone). Patients harboring this genotype were overrepresented in St George Central, the capital city of Port-of-Spain, younger (mean 39.1 years vs. 47.7 years for other genotypes, p<0.0005), and more frequently prison inmates and drug users, while those harboring “other genotypes” were older and showed diabetes as an associated factor. A study of the evolutionary relationships suggested probable relatedness of SIT566 with X1 prototype SIT119. A database search localized most of the SIT566 related patterns in USA. Second-line typing using Mycobacterial Interspersed Repetitive Units (MIRUs) suggested the highly conserved nature of SIT566, its phylogeographically specificity to T&T.

Acknowledgements We thank the Caribbean Epidemiology Center, Trinidad & Tobago for support, and the staff of the Chest Clinics at the Eric Williams Medical Sciences Complex, the San Fernando General Hospital, and Trinidad Public Health Laboratory for assistance with data collection. SB is grateful to the University of West Indies for financial support, and JM to the Regional Council of Guadeloupe and European Social Funds for a Ph.D. fellowship. NR and JM thank J. Driscoll for providing information regarding the SIT566 clone in USA.

78 ESM 2009 PP-6

SPOLIGOTYPE PATTERNS AND DRUG RESISTANT PROFILE OF Mycobacterium tuberculosis IN SUDAN

Ghada Suliman Sharaf-Eldin 1, Imad F.Elmoula 1, Mohammed S. Ali 1, Nageeb S. Saaed 2, Ahammed B Ali 1, Kim Mallard 3, Ruth McNerney 3, Saad Algamdi 3 1 - Al Neelain University-Sudan 2 - National Health Laboratory-Sudan 3 - London School of Hygiene & Tropical Medicine.

Sudan has a high burden of tuberculosis with an estimated 93,000 new cases each year. The purpose of this study was to investigate the genotypic patterns of M. tuberculosis strains circulating in Sudan and to assess their susceptibly to anti-tuberculosis drugs. Isolates from 237 smear positive tuberculosis patients were collected from different geographic regions of the country. Spoligotyping was performed by the Kamerbeek method and results were compared with the international SpolDB4 database (Institut Pasteur, Guadeloupe). Results revealed 28 clusters ranging in size from 12 to 57 isolates. Seventy unique (unclustered) strains were observed, representing 30% of the strains examined. The most frequently observed spoligotype patterns belonged to the CAS family which represented 115 (48.5%) of isolates studied. T1, H3, U and Beijing strains were found in 12 (5.1%), 11 (4.6%), 7 (3%) and 6 (2.5%) patients respectively. Strains belonging to the Beijing family were found mainly in Western Sudan. Resistance to isoniazid, rifampicin, ethambutol and streptomycin was observed in 18.1, 22.4, 22.2 and 32% of strains respectively. Twenty patients (8.4%) had MDR-TB of which 10 were new cases. Seventeen patients with rifampicin resistant tuberculosis were infected with CAS1-DELHI strains matching SIT 25 of the SpolDB4 database and 3 were of the SIT 1 Beijing family. 15 loci MIRU-VNTR typing subdivided the 17 CAS strains into one cluster of 5, two clusters of 2 and 8 individual MIRU types. Similarly the 3 Beijing spoligotypes were differentiated into a cluster of 2 and a single strain. The use of molecular strain typing provides a proactive approach that may be used to initiate, and not just augment, traditional surveillance outbreak investigation in Sudan. However, caution must be used when interpreting clustered spoligotype patterns in this region.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 79 PP-7

CONTROVERSIAL DISSEMINATION PATTERN OF THE BULGARIA -SPECIFIC M. tuberculosis SPOLIGOTYPE ST125_BGR

Violeta Valcheva 1, 2, Igor Mokrousov 1, 3, Stefan Panaiotov 4, Elizabeta Bachiiska 4, Thierry Zozio 1, Christophe Sola 1, Nadya Markova 2, Nalin Rastogi 1 1 - Institut Pasteur de Guadeloupe, France 2 - Institute of Microbiology, Sofia, Bulgaria 3 - St. Petersburg Pasteur Institute, St. Petersburg, Russia 4 - National Center of Infectious and Parasitic Diseases, Sofia, Bulgaria

Objective To investigate phylogenetic position and geographic genetic diversity of spoligotype ST125 in Bulgaria.

Material and Methods Study sample included all available 47 DNA samples belonging to spoligotype ST125 that were taken from two previously published M. tuberculosis collections from Bulgaria (Valcheva et al., 2005, 2007, 2008abc; Panaiotov et al., 2005, 2006). These DNA were additionally typed using new 24-loci MIRU format, IS6110-RFLP typing and LAM-PCR. Phylogenetic analysis was done using PAUP and PHYLIP packages.

Results Comparison with SITVIT2 database (Institut Pasteur de Guadeloupe) revealed a high gradient of ST125 in Bulgaria (14.3%) compared to its negligible presence in the world. Typing of Bulgarian ST125 strains revealed that they: (i) did not harbor a LAM-specific IS6110 insertion (ii) formed a monophyletic cluster in 24-MIRU tree of Bulgarian strains (iii) grouped closely with ST34 that is a prototype of the S family. A similarity of the IS6110-RFLP profiles confirmed a true relatedness of these ST125 strains whereas a diversity of the MIRU loci suggested a long-term evolution of this spoligotype in Bulgaria. Minimum spanning tree of the 24-MIRU-based subtypes of ST125, and comparison with their geographic distribution revealed an enigmatic and complex dissemination pattern of this spoligotype across Bulgaria.

Conclusion T125 likely belongs to S family and may have originated from spoligotype ST4 by a deletion of a single spacer #40 in the DR locus. ST125 is phylogeographically specific for Bulgaria; we propose its renaming as ST125_BGR. A high diversity of the MIRU loci suggests a long-term evolution of this spoligotype in Bulgaria.

Acknowledgments. This work was supported by NATO grant SFP-982319 “Detect drug-resistant TB”.

80 ESM 2009 PP-8

Mycobacterium tuberculosis BEIJING GENOTYPE AND ORIGINS OF THE BULGARIANS

Panaiotov, Stefan; Bachiyska, Elizabeta; Brankova, Nadia; Levterova, Victoria National Center of Infectious and Parasitic Diseases, Sofia 1504, Bulgaria

Recent studies demonstrated that exist genotypes of M. tuberculosis locally distributed to specific geographic region. Other genotypes are distributed globally or on vast geographic areas. These facts led to other recent fundamental studies and conclusions that exists certain genetic predisposition of the ethnic groups to specific M. tuberculosis genotypes, hence genetic predisposition to tuberculosis specific genotypes is suspected. In the past and nowadays the waves of human migration coincide with expansion of tuberculosis genotypes. In our study we associated the global distribution of MTB Beijing genotype, its’ distribution in Bulgaria, the geographic regions of historical origin of the Bulgarian tribes, and the ethnic affiliation of the Bulgarians. For our analysis we used data published in the fourth international spoligotyping database (SpolDB4), publications, personal communications and official historical theories regarding origins of the Bulgarians. From the literature and in SpolDB4 exists about 330 spoligotypes of Bulgarian M. tuberculosis clinical strains collected from all over the country. Beijing spoligotype was not identified among them. We concluded that this MTB genotype is not (or very rare) distributed in Bulgaria. In Romania Beijing genotype was not identified too (personal communication). Other countries associated with the origins and migration of the Bulgarians where Beijing genotype is not identified is Iran. Significant part of the Bulgarian spoligotypes are phylogenetically related to MANU family, widely distributed in India. These facts correlate with the widely accepted theory that the origins of the Bulgarian tribes are of Indo-Iranian lineage. In contrary, this fact does not support the theory for the Turkik lineage or more precisely the Turano-Hunnic origins of the Bulgarian ethnos. Beiging genotype is widely distributed in Central Asian countries, Kazahstan, Turkmenistan, Russia, Turkey, China etc. Bulgaria has very active tourist, cultural, political, trade and immigration links with all these countries. Based on these empiric facts we indirectly conclude that there is genetic predisposed resistance of the Bulgarian and Iranian ethnos to MTB Beijing genotype.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 81 PP-10

THE EXTENT OF THE LATIN AMERICAN-MEDITERRANEAN Mycobacterium tuberculosis SPOLIGOTYPE FAMILY IN PORTUGAL

Suzana David 1, João Nuno Ribeiro 1,2, José-Nuno Maio 1,2, Inês João 1, António Amorim 3, Edna Pereira 3 1 - Unidade de Referência e Vigilância Epidemiológica, Laboratório de Infecções Respiratórias – Micobactérias, Instituto Nacional de Saúde Dr. Ricardo Jorge, Portugal 2 - Grupo de Microbiologia e Imunologia da Infecção, Instituto de Biologia Molecular e Celular, Porto, Portugal 3 - Laboratório de Saúde Pública, Micobacteriologia / Tuberculose, Administração Regional de Lisboa e Vale do Tejo, Lisboa, Portugal

Portugal is classified at the intermediate level with respect to the incidence for tuberculosis. Geographical spread is heterogeneous with the majority of cases concentrated in the large urban areas mainly in the Lisbon and Porto districts. Previous efforts in the characterization of the population structure of Mycobacterium tuberculosis isolates stressed the importance of this approach to tuberculosis control. Spoligotyping data restricted to the metropolitan area of Lisbon, revealed a 51% prevalence of the Latin American-Mediterranean (LAM) spoligotype family, and a high proportion of SIT20 (LAM1) and SIT42 (LAM9) sub-families. In the present study this characterization has been extended to encompass both the Lisbon and Porto areas. With the use of complementary data from SpotClust and Ag85C103 RFLP, the proportion of the LAM genotype was shown to be as high as 62% of the total number of isolates. The LAM genotypes were further characterized for the RDRio deletion detected in up to 60% of the LAM isolates. This study suggests that Portugal may have one of the highest global proportions of the LAM family. Monitoring and further characterizing of this relevant spoligotype family is considered of major importance for the understanding of the dynamics of tuberculosis in Portugal.

82 ESM 2009 PP-11

FITNESS STUDY OF THE RDRIO LINEAGE AND LAM FAMILY OF Mycobacterium tuberculosis IN A STUDY POPULATION IN RIO GRANDE, BRAZIL

Von Groll, Andrea 1; Martin, Anandi 1; Felix, Carolina 2; Prata, Pedro 2; Honscha, Günther 3; Portaels, Françoise 1; Almeida da Silva, Pedro 2; Palomino, Juan Carlos 1 1 - Institute of Tropical Medicine, Antwerp, Belgium 2 - Universidade Federal do Rio Grande, Rio Grande, Brazil 3 - Laboratório Municipal de Tisiologia, Rio Grande, Brazil

RDRio is a novel Mycobacterium tuberculosis lineage member of the Latin American-Mediterranean (LAM) family. LAM has been found worldwide but it is more predominant in South America. The aim of this study was to assess the presence of the RDRio lineage and LAM family in the city of Rio Grande, Brazil, and to investigate the fitness of these strains based on the determination of their rate of growth. Fifty clinical isolates of M. tuberculosis were genotyped and 43 different patterns were found by spoligotyping and MIRU-VNTR. The predominant genotypes belonged to the LAM family (54% of the strains) followed by clade T (22%) and Haarlem (16%). The RDRio lineage represented 38% of the total strains and 70.4% of the LAM strains found in this study. Strains belonging to the LAM family showed a fitness advantage comparing their rate of growth with that of non-LAM. RDRio strains were predominant within the LAM family, but a significant difference in fitness between RDRio and the non-RDRio strains was not confirmed.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 83 PP-12

Genomic characterization of Lisboa family strains by deletion analysis

João Perdigão, Carla Silva and Isabel Portugal Centro de Patogénese Molecular, URIA, Faculdade de Farmácia da Universidade de Lisboa

Multidrug and extensive drug resistant tuberculosis poses a very serious threat for public health. Lisbon Health Region has one of the world’s most serious situations regarding this problem. Such, is the result of a continued circulation of an endemic and predominant strains of a particular genetic family – Lisboa family. Little is known regarding the phylogeny, relative virulence and genetic background of these strains. The loss or deletion of specific genomic regions constitutes the most important way by which Mycobacterium tuberculosis diverges and adapts. Several deletions, named Regions of Difference (RD), have already been described and associated with phylogeographic lineages. The characterization of Lisboa family in this manner may elucidate its origin and perhaps be helpful in explaining its high prevalence. Three representative clinical isolates of different genetic clusters of strains circulating in Lisbon Health Region were screened for the presence of 16 distinct RDs. Deletion detection was performed by PCR carried out using primers flanking each RD. Confirmation was performed by sequencing analysis. All three isolates were found to possess four of the tested deletions: TbD1, pks15/1, RD174 and RDRIO. It was not possible to discriminate between the strains using this deletion typing approach. However, it was possible to infer on the phylogeography of these strains. The presence of TbD1 and pks15/1 deletion positions the strains in the modern and Euro-American lineage, respectively. On the other hand, RD174 suggests that the analyzed strains are related to the West-African sub-lineage. The present study point toward the fact that Lisboa strains and others circulating in Lisbon belong to Mycobacterium tuberculosis modern lineages of the Euro-American lineage, West-African sub-lineage, therefore revealing more on Lisboa family’s origin.

84 ESM 2009 PP-13

IMPORTANCE OF MOLECULAR TYPING IN SUSPECTED INTRA-FAMILIAL TRANSMISSION OF TUBERCULOSIS

Obrovac, Mihaela 1, Katalinic-Jankovic, Vera 1, Grce, Magdalena 2, Zmak, Ljiljana 1 1 - Croatian National Institute of Public Health 2 - Rudjer Boskovic Institute

Tuberculosis (TB) is most commonly transmitted from a person suffering from infectious pulmonary TB to another person by infected droplet nuclei. The chance that a person will be infected with TB depends on the intensity, frequency and duration of the exposure to tubercle bacilli. Also, numerous studies emphasize the importance of host resistance and hereditary susceptibility, indicating that the development of TB is a result of a complex interaction between the host and the pathogen influenced by environmental factors. Assuming that there is prolonged duration and frequency of contact between family members and among household contacts, it is estimated that the risk of TB transmission will be high. There are several studies confirming intra-familial transmission using genotyping of isolated M. tuberculosis strains. However, the possibility of unsuspected transmission should not be disregarded. We report here of two cases of suspected TB transmission in families caused by M. tuberculosis strains that were found not to be identical according to genotyping profiles obtained by determining variabile number of tandem repeats (VNTR) of mycobacterial repetitive interspersed units (MIRU). Genotyping was performed using complete set of 24 VNTR loci. Epidemiological data were collected by contact tracing and interviewing patients. Our results show that TB cases in family do not necessarily have to be caused by the same M. tuberculosis strain. Epidemiologic investigations need to be combined with genotyping data for better understanding of transmission dynamics. Transmission of TB cannot be confirmed by contact investigation only, even when intra-familial transmission is suspected.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 85 PP-14

DETECTION OF CLONAL COMPLEXITY IN CLINICAL M. tuberculosis ISOLATES BY MIRU-VNTR IN CUKUROVA REGION, TURKEY

Ülkü ORAL ZEYTINLI, M. Begüm KAYAR, Ayse KARACALI, Arzu SAHAN KIPALEVErkan YULA, Fatih KÖKSAL University of Cukurova

Purpose of the study: Tuberculosis remains one of the most prevalent infectious disease in the world . The application of molecular typing methods to the analysis of clinical Mycobacterium tuberculosis (MTB) complex isolates has greatly facilitated the understanding of epidemiology of tuberculosis (TB) and revealed that the infection by this pathogen can be clonally complex and reinfection, coinfection. Genotyping using RFLP-IS6110 (Restriction Fragment Length Polymorphism) is based on transposable element IS6110 and mycobacterial interspersed repetitive unit-variable number of tandem repeat typing (MIRU-VNTR) has become a major method for epidemiological tracking of Mycobacterium tuberculosis complex clones. Our aim was to establish the range of applicability of 12 loci MIRU–VNTR genotyping in epidemiology of TB and evaluate the discriminatory power obtained with RFLP-IS6110 and MIRU-VNTR used alone or in combination.

Methods In this study, we analyzed 94 clinical MTB complex isolates from sputum in patients with pulmonary TB between February 2008- February 2009 in Cukurova region, Turkey.

Results MIRU–VNTR typing detected 45 different patterns, 61 strains were grouped into 12 clusters and 33 strains had uniqe patterns. The largest cluster comprised 9 strains. In addition, 2 clusters contained 5 strains, 6 clusters contained 3 strains and 1 cluster contained 2 strains. It is also determined that the loci including MIRU 04, MIRU 10, MIRU 26 and MIRU 40 have the highest allelic diversities and discriminatory power. On the other hand with IS6110-RFLP typing of same clinical MTB strains, 33 genotypes were founded. 76 strains of which were gruoped into15 clusters. 18 of the 94 strains had unigue RFLP patterns.

Conclusion MIRU-VNTR is more discriminative methods for phylogenetic studies than IS6110-RFLP and could define from reinfections to reactivations important to treatment of MTB complex organisms.

86 ESM 2009 PP-15

MOLECULAR EPIDEMIOLOGY STUDY OF TUBERCULOSIS PATIENTS IN A SMALL CITY OF SÃO PAULO – BRAZIL, FROM 2002 TO 2006

Leite, Clarice; Santos, Adolfo; Pandolfi, José Rodrigo; Malaspina, Ana C; Pavan, Fernando; Mendes, Natália Universidade Estadual Paulista, Faculdade de Ciências Farmacêuticas

The molecular epidemiology study using different techniques revolutionized the understanding of the epidemiology of tuberculosis allowing comparison between strains of Mycobacterium tuberculosis and tracking the movements of individual lines. This project aimed to use the techniques of molecular epidemiology (MIRU) trying to understand more about the phenomenon of transmission of tuberculosis among patients with pulmonary tuberculosis in a small city of São Paulo - Brazil, attended the Special Health Service of Araraquara (SESA - clinic of reference in the diagnosis and treatment of tuberculosis) in the city of Araraquara. From total 163 positive cultures received, the MIRU technique was performed in 74,2% (121/163) of the isolates from patients attended by SESA in the period from 2002 to 2006. 5 isolates were also identified as environmental mycobacteria and 4 unidentified mycobacteria. From the 121 isolates submitted to genotyping, six did not present all alleles among the 12 loci of MIRU. From the 115 isolates submitted to the dendrogram, 29 (25,2%) are grouped into 13 clonal groups with similarity of 100%. 10 groups with two isolates each and 3 groups containing 3 isolates each. The others 86 isolates (74,8%) had a single genetic profile. About the group of 29 patients, only 10 were female and 19 males. Except for one patient, the other 28 were treated with the schedule I having cure in 82,8% of the cases. Whereas the similarity of 83% or greater, highlighted 3 major clonal groups called A, B and C, involving 86 of all isolates analyzed. In these large groups were included all 13 clonal groups with 100% similarity. These data suggest the possibility the tuberculosis in Araraquara is because of the presence of persistent endemic strains responsable for 74,8% of cases, besides the existence of recent transmission in this work was 25,2%.

Keywords: Epidemiology, Tuberculosis, MIRU Financial support: FUNDUNESP.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 87 PP-16

GENOTYPING OF Mycobacterium tuberculosis IN NORTHWEST OF PARANÁ STATE OF BRAZIL

Leite, Clarice Queico Fujimura 1, Nogutia, Erika N 2, Malaspina, Ana Carolina 1, Santos, Adolfo Carlos Barreto 1, Hirata, Rosáro DC 3, Hirata, Mário H 3, Cardoso, Rosilene Fressatti 2 1 - São Paulo State University 2 - Maringá State University 3 - University of São Paulo

Introduction Worldwide, tuberculosis (TB) remains the most frequent and important infectious disease causing morbidity and death. The molecular epidemiology study using different techniques revolutionized the understanding of the epidemiology of tuberculosis allowing comparison between strains of Mycobacterium tuberculosis and tracking the movements of individual lines. We reported here the first insight about the genetic diversity of M. tuberculosis in northwest of Paraná State, south of Brazil. This knowledge encourages additional prospective epidemiological study for evaluation of the Regional Tuberculosis Control Plan in this setting.

Objectives Provide information about the genetic diversity and prevalent genotype of Mycobacterium tuberculosis and compare the usefully of two methodologies in epidemiological study of tuberculosis in low endemic area in south of Brazil. Material and Methods: We used spoligotyping and MIRU-VNTR typing to genotype M. tuberculosis isolates.

Results The 93 isolates analyzed by spoligotyping were divided into 36 different patterns and 25 were described in the SpolDB4.0 database. Latin American and Mediterranean, Haarlem and T family were responsible for 26.9%, 17.2% and 11.8%, of tuberculosis cases respectively. From the 84 isolates analyzed by MIRU-VNTR typing, 58 showed unique pattern and 26 belonged to 9 clusters. The MIRU loci 40, 23, 10 and 16 were the most discriminatory. MIRU-VNTR and spoligotyping combined showed 85.7% of discriminatory power (HGI=0.995).

Conclusions Spoligotyping and MIRU-VNTR typing combined are useful tool for epidemiological study in this low endemic setting in south of Brazil and tuberculosis predominantly develops through reactivation of latent infection.

88 ESM 2009 PP-17

SPOLIGOTYPING OF Mycobacterium tuberculosis ISOLATED FROM PATIENTS OF CLEMENTE FERREIRA AMBULATORY IN SÃO PAULO, SP – BRAZIL

Mello, Fernado Augusto Fiuza 1, Albarral, Maria Idemar Pedrosa 1, Mendes, Natália Helena 2, Pandolfi, José Rodrigo Cláudio 2, Santos, Adolfo Carlos Barreto 2, Almeida, Elisabete Aparecida 1, Cardoso, Rosilene Fressatti 3, Leite, Clarice Queico Fujimura 2 1 - Clemente Ferreira Institute 2 - São Paulo State University 3 - Maringá State University

The molecular epidemiology study using different techniques revolutionized the understanding of the epidemiology of tuberculosis allowing comparison between strains of Mycobacterium tuberculosis and tracking the movement of individual strains. This project aims to use the technique of molecular epidemiology, Spoligotyping, trying to understand more about the phenomenon of transmission in patients with pulmonary tuberculosis treated at Clemente Ferreira Ambulatory (ambulatory of reference for the treatment of tuberculosis) in São Paulo city, from August 2006 to July 2008. The clinical isolates were re-identified by molecular technique (PCR and PRA), and the strains identified as M. tuberculosis conducted by the genotyping technique of Spoligotyping. From 102 isolates, the technique of IS 6110-PCR confirmed the identification of M. tuberculosis in 96 clinical isolates and the PRA in 99, the remaining 3, 2 isolates identified as M. avium subtype 2 and 1 unidentified mycobacteria. The results showed that 3 clinical isolates of M. tuberculosis had not the IS6110 insertion sequence specific of M. tuberculosis, as well as 3 isolates identified in the clinic as M. tuberculosis by molecular techniques were atypical mycobacteria. Of 96 isolates confirmed as M. tuberculosis, were analyzed by the technique of Spoligotyping, a total of 89 isolates, which revealed the presence of 21 strains (23.6%) with spoligotipes not yet described in the data base world (spolDB4) and 68 (76.4%) of isolates involved in 7 different families, containing 2 to 30 isolates. The most frequent was T family with 30 isolates), followed by LAM (with 20 isolates) and Haarlem (with 10 isolates), which together accounted about 67.4% of all isolates. Were also identified 4 genotypes of the Beijing family, all simultaneously resistant to isoniazid and rifampicin and / or more drugs.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 89 PP-18

COMPARISON OF Mycobacterium BEIJING GENOTYPE WITH VNTR, SPOLIGOTYPING AND RFLP-IS6110

Elahe Tajeddin , Parissa Farnia, Mohammad Kargar,Jamileh Noroozi, Mojtaba ahmadi, Mehdi kazempour, Maryam Hadadi,Mohammadreza Masjedi, Aliakbar Velayati Mycobacteriology Research Center (MRC) National Research Institute Of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University Medical Campus.Tehran,Iran.

Background Beijing strains constitute more than 1/4 of Mycobacterium tuberculosis (MTB) genotypes. Beijing genotype is considered an important genotype because of its reasonable characteristics such as: association with multi-drugs resistance TB. Accordingly these strains are reluctant to conventional TB drugs. Therefore, it is necessary to investigate the transmission rate among Beijing strains within the studied communities. In this study, three molecular methods (Spoligotyping, VNTR, and RFLP-IS6110) were used to identify transmission among patients infected with Beijing strains.

Materials and Methods The susceptibility tests were performed on 238 M. tuberculosis culture positive specimens. Thereafter, the isolated Beijing genotype was subjected to VNTR and RFLP. The results of Spoligotyping were analysed by using SPOLDB4 database. VNTR typing was used to identify alleles diversity in 9 locus (MPTR-A, ETR-A, ETR-B, ETR-C, ETR-D, ETR-E, ETR-F, QUB11B, QUB3232) of isolated Beijing strains.The allelic diversity of VNTR was measured by using Hunter Gaston Index (HGI).

Results The spoligotyping of M. tuberculosis isolates revealed the following 8 patterns: Haarlem (27.7%), CAS1 (25.2%), EAI3 (21.8%), CAS2 (6.7%), T1 (6.3%), Beijing(5.5%) U(5%), T(0.4), EAI2 (1.2%). The following VNTR loci (QUB3232), (QUB11b, ETR-E and ETR-F) and (other loci) were identified as most (HGI≥ 0.6), median (HGI≥0.4-0.6) and weakest (HGI=0) distinctive loci for Beijing families respectively. Whereas the Beijing strains demonstrated diverse patterns in RFLP,13/13(100%) and VNTR 10/13(77%).

Conclusions Beijing is one of the dominated circulating strains in Iran and interestingly majority of infected cases were due to reactivation rather than recent transmission.The VNTR and spoligotiping methods were more efficient to detect Beijing strains than by use VNTR and RFLP allow.

Keywords Spoligotyping / VNTR / RFLP / Mycobacterium tuberculosis Beijing genotype.

90 ESM 2009 PP-19

MULTIPLY RECURRENT TUBERCULOSIS IN A PACIENT LIVING WITH HIV: REINFECTION OR REACTIVATION?

Ritacco, Viviana 1, Reniero, Ana 2, Beltrán, Marcelo 2, López, Beatriz 1, Kantor, Isabel 3, Barrera, Lucía 1 1 - ANLIS, CONICET, Argentina 2 - Hospital Municipal de San Isidro 3 - PAHO/WHO consultant

Purpose To determine the cause of recurrent clinical episodes of tuberculosis in a patient living with HIV. Patient: Male, 24 year-old and illegal drug intravenous user for 10 years at the time of first consultation, assisted between 1995 and 2009 in our outpatient clinic, San Isidro Municipal Hospital, Argentina.

Methods Clinical-epidemiological follow up. Mycobacterial culture, identification and drug susceptibility testing. Mycobacterium tuberculosis IS6110 RFLP and spoligo genotyping within the frame of a population-based study performed in San Isidro, an outskirt of Buenos Aires City.

Findings Our patient’s first diagnosis of tuberculosis was confirmed by culture in 1995, almost simultaneously with HIV serological conversion. At that time, the isolate was found only resistant to isoniazid and its genotype matched that of a isoniazid- resistant outbreak strain previously identified in two of our patient’s prison inmates, who were also members of his intravenous-drug-user gang. One year after cure, our patient suffered from a relapse of his tuberculosis due to the same strain, which now had added resistance to rifampicin and lost a band in the IS6110 fingerprint. In 2002, the patient suffered from a third episode of tuberculosis, this time due to a fully drug susceptible strain, identified previously in other of our patient’s intravenous drug user friends. At that time, given his poor clinical condition, our patient underwent a prolonged hospitalization in the Muñiz Hospital, the epicentre of a large tuberculosis outbreak caused by the notorious multidrug-resistant strain “M”. After successful treatment and cure, our patient, who was never compliant with antiretro- viral treatment, was discharged from the Muñiz Hospital. A few months later, he sought again assistance at our clinic with active tuberculosis due to the multidrug resistant strain “M”. He is currently struggling with a relapse of disease caused by this outbreak strain.

Conclusion Whether recurrent tuberculosis is due to a newly acquired infection or to reactivation of a previous one is a century- long controversial question. In our case, both conditions alternated throughout the 14 years of the patient living with HIV. Work partially funded by FP7 grants 201690 and 223373 of the EC.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 91 PP-20

MOLECULAR EPIDEMIOLOGY OF TUBERCULosis IN VILA NOVA DE GAIA, PORTUGAL

Tavares Magalhães, Ana1, Alves, Adriana1, Braga, Rosário2, Valente, Isabel2, Duarte, Raquel3 and Miranda, Anabela1 1 - Tuberculosis Reference Laboratory, Department of Infectious Diseases, National Institute of Health, Porto, Portugal 2 - Central Hospital, Vila Nova de Gaia, Portugal 3 - Chest Clinic, Vila Nova de Gaia, Portugal

DNA fingerprinting of Mycobacterium tuberculosis ��has provided a better understanding of the epidemiology of tuberculosis. Restriction fragment length polymorphism based on IS6110 (RFLP-IS6110) has been the gold standard for typing M. tuberculosis since 1993. In recent years, mycobacterial interspersed repetitive units variable number tandem repeat (MIRU-VNTR) has been proposed as a first-line typing method for M. tuberculosis. This technique generates easily analyzed and portable data, has a good discrimination power and has been proven useful for studying the epidemiology of tuberculosis and the phylogeography of tuberculosis bacilli. In the present study we evaluated the genetic diversity of M. tuberculosis clinical strains, isolated from 115 patients from Vila Nova de Gaia, Portugal, during the period of 2004 to 2005, using the standardized MIRU-VNTR typing method based on 15 loci, proposed by Supply and collaborators in 2006. Strain lineage designation, allelic diversity and clustering rate were determined using the MIRU-VNTRplus identification database. The discriminative power of the method was analysed using the Hunter and Gaston diversity index. Based on MIRU-VNTR typing, the 115 strains were divided into 62 types as follows: 69 strains were distributed into 16 clusters containing two to eight isolates, and 46 strains had unique profiles. MIRU-VNTR revealed a clustering rate of 46% in the sample under study. The Hunter-Gaston index was 0.976. The most discriminatory loci were Mtub04, MIRU 40, MIRU10, QUB11b, Mtub30, Mtub39 and QUB26 showing an allelic diversity higher than 0,6. The phylogenetic analysis revealed the presence of two major lineages, LAM and Haarlem, representing 60% and 27% of the total isolates, respectively. This fact is in agreement with the M. tuberculosis phylogeography for the South of Europe. Fourteen strains showed drug resistance but no association was established between drug resistance profile and phylogenetic family. MIRU-VNTR showed a good discriminatory power for typing of M. tuberculosis strains in this setting and the MIRU- VNTRplus database is an important tool for lineage identifi cation. This typing approach offers, simultaneously, epidemio-is an important tool for lineage identifi cation. This typing approach offers, simultaneously, epidemio-lineage identification. This typing approach offers, simultaneously, epidemiologic and phylogenetic information, as demonstrated. Overall, this study reveals that recent transmission of tuberculosis is high in Vila Nova de Gaia, and that import of strains in this region is not a problem.

92 ESM 2009 PP-21

MOLECULAR STUDY OF RECURRENT TUBERCULOSIS CASES

Alves, Adriana; Miranda, Anabela; Tuberculosis Reference Laboratory Group Tuberculosis Reference Laboratory, Department of Infectious Diseases, National Institute of Health, Porto, Portugal

Tuberculosis recurrence is frequently attributed to reactivation of the isolate responsible for the first episode of the disease. Nevertheless, this can also be due to an infection with another isolate, or to mixed infections. Clarification of the cause of recurrence is very important and can be achieved by molecular typing of serial isolates of M. tuberculosis. The most appropriate methods to do so are IS6110 RFLP and MIRU-VNTR. In this work, 39 clinical isolates of M. tuberculosis belonging to nine individuals with recurrent disease were studied throughout time. Seven of these patients were resistant to isoniazid and rifampicin as well as to most other 1st and 2nd line drugs. Another patient was resistant to isoniazid and streptomycin, and the last one was resistant to rifampicin only. For some of these individuals, resistance to drugs worsened during the cause of the disease, which in some cases has lasted for more than a decade. All 39 strains were analyzed by IS6110 RFLP. MIRU-VNTR was used to type: (i) the first strain of each patient when serial isolates showed no change in the IS6110 RFLP profile; or (ii) each strain with a pattern change in relation to the previous one. RFLP results showed that only one out of nine patients displayed a change in the pattern of serial isolates with gain of one IS6110 element. Analysis of the results using Bionumerics grouped these patients in nine clusters, being that: (i) strains from two patients belong to the same cluster; and (ii) strains of one patient are divided in two clusters. Results of 15 MIRU-VNTR loci typing corroborate the IS6110 RFLP findings. In other words, the serial isolate that gained one IS6110 element also shows a change in MIRU-VNTR results. In this case, we observed a reduction of one repeat in loci Mtub21 and QUB11b. Finally, 40% of these strains belong to the LAM family, 10% to the Haarlem family, and the remaining 50% did not find a match in the database. This work shows that the cause of recurrent tuberculosis of the nine patients included in this study is due to persistence of initial M. tuberculosis strain or to reactivation when there is more than one episode of disease. Antibiotic resistance is the most important cause of chronic tuberculosis in these patients, since all analyzed strains are resistant to the majority of the 1st and 2nd line drugs.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 93 PP-22

GENOTYPING OF DRUG RESISTANCE IN Mycobacterium tuberculosis USING DIAGNOSTIC MICROARRAYS

Ehricht, Ralf 1, Slickers, Peter 1, Monecke, Stefan 2 1 - CLONDIAG, Jena, Germany 2 - University Hospital Dresden

Tuberculosis is a disease of worldwide concern. Antimicrobial resistance in Mycobacterium tuberculosis is an increasing challenge and, in contrast to other bacteria, not caused by the acquisition of certain genes, but by acquisition of single point mutations in genes which are present in all strains. Unfortunately, culturing and subsequent growth inhibition assays are still time demanding preventing fast detection and treatment. Genotyping methods as PCR followed by sequencing are an alternative. Here the bottlenecks are processing time, overall costs and lack of parallelisation. Hybridisation of such PCR products against highly discriminatory oligonucleotide probes is an alternative approach which could solve these problems. We developed an assay using a diagnostic oligonucleotide microarray and covering probes for relevant mutations in genes rpoB, katG, embA, and embB, for the embC/embA-intergenic region, and the mabA/inhA promoter. PCR is employed to amplify these genes and to incorporate biotin 16-dUTP during elongation. These labeled amplicons are hybridised to the array which are inserted into ArrayStrips, and hybridisation is visualised using dye precipitation trig- gered by streptavidin-peroxidase complexes bound to the biotin labels and the ArrayMate reading device. The procedure is currently tested using DNA from previously characterized strains for which conventional susceptibility test results and relevant sequences are available.

94 ESM 2009 PP-23

GENOTYPING OF MONO AND MULTI-DRUG RESISTANCE TB IN SAUDI ARABIA

Sahal Al-Hajoj1, Bright Varghese1, Mais Herbawi1 , Ruba Al-Omari1 and Caroline Allix-Béguec2 1 - King Faisal Specialist Hospital and Research Centre, Comparative Medicine Tuberculosis Research Unit Saudia Arabia 2 - Genoscreen

A total of 150 isolates collected from different regions in Saudia Arabia were the subject of finger printing using GenoScreen MIRU-VNTR kit (do you know the timeframe?). Upon genotype the data were compared to the international MIRU- VNTRplus database (www. Miru-vntrplus.org). The data showed that Saudia Arabia harbors the following major clades EAI 13.07%, Haarlem 12.42%, TUR 13.07%, Beijing 12.42%, unknown12.42%, Dehli/CAS 7.84%, LAM 7.19%, Cameroon 6.54%, UgandaI 5.23%, S 3.92%, multiple matches1.96%, NEW-1 1.31%, URAL 0.65%, Ghana 0.65%, X, 0.65%, UgandaII, 0.65%. Indicate the clustering rate for this panel of 150 isolates. Ongoing transmission may be an indication to the need of improving TB control program. The unknown clades represent a considerable % (12.42%). These could represent unique endogenous clades. However, further analysis is needed to gain insight into the nature of the unknown clades.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 95 PP-24

early detection of MDRtb by molecular tools in the control of drug resistant tuberculosis in portugal: a case of success

Diana Machado1, Miguel Viveiros1,2, Liliana Rodrigues1,3, Isabel Couto1,4, Leonard Amaral1,2,3 1 - Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Lisbon, Portugal 2 - COST ACTION BM0701 (ATENS) 3 - UPMM, IHMT/UNL, Lisbon, Portugal 4 - Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e Tecnologia, UNL, Caparica, Portugal

Multidrug resistant tuberculosis (MDRTB) represents a threat to public health and a challenge to tuberculosis (TB) control programs. From 1994 to 1997, Portugal had an incidence of 48.3 new cases of TB per 100 000 inhabitants and an average of 22.7% of these cases were MDRTB, the highest in Western Europe. In an attempt to assist the National Health Authorities in the control of TB, we implemented in 2002 with the support of Fundação Calouste Gulbenkian, the “TB Fast-Track” program as part of the TB Task Force of Greater Lisbon, involving 12 Lisbon hospitals and based on the use of the BACTECTM MGIT 960 system, coupled with the direct identification of M. tuberculosis complex (MTBC) and the detection of mutations in the rpoB gene, using the INNOLiPA Rif.TB Assay (LiPA) (Innogenetics). Because mutations in the rpoB result in resistance to rifampicin (RIF), and resistance to RIF is almost always accompanied by resistance to isoniazid (INH), this approach allowed us to identify the MDRTB patient within 24-48 hours. A full report confirming identification and antibiotic susceptibility (AST) of MTBC by conventional methods (BACTEC culture plus AST and Accuprobe ID) was issued within additional 12 days.

From September 2002 to January 2006, the LiPA assay was directly applied to 630 acid fast positive respiratory specimens. The comparison between data from this assay and conventional methods revealed 84 discrepancies. The 11 false positive results corresponded to patients with therapy already established by the time of specimen sampling, whereas the 73 false negative resulted from inhibition of amplification. A total of 487 of the 600 MTBC positive isolates were susceptible to all 5 first-line anti-TB drugs. The frequency of MDRTB (resistant to at least INH plus RIF) was 10%. From the 63 MTBC resistant to RIF, 62 were detected by the LiPA as carrying mutations S531L (60 isolates), H526Y and D516V (1 isolate each). No mutation was detected by LiPA for one sample, repeatedly identified as resistant by AST. Detection of rpoB mutations proved to be a good surrogate marker for MDRTB, since only 2 out of 600 MTBC isolates were monoresistant to RIF.

The early detection of active TB, particularly the detection of MDRTB, is essential for the success of any TB control program. The application of molecular techniques for the early identification of MDRTB assisted the National Health Authorities in the reduction of MDRTB rates in Lisbon to less than 8% (average 2003 to 2007).

96 ESM 2009 PP-25

Drug-resistance of Mycobacterium tuberculosis at penitentiary institutions of St. Petersburg, Russian Federation.

Vladimirov, Kirill; Zaitseva, Elena; Ivanov, Aleksandr Institution: State Medical Academy named after I.I. Mechnikov, St. Petersburg, Russia.

Background Morbidity with tuberculosis (TB) in Russian Federation and the whole world remains high. This index is up to 40 times above the average level among prison population, with high prevalence of multi-drug resistant (MDR) TB. Setting. Central hospital for prisoners in St. Petersburg. Study design. We retrospectively reviewed data of the patients, who were admitted to the hospital for active culture- positive TB between 2005 and 2008. Between 2005 and 2007, new and re-treatment cases were admitted. In 2008, only new TB cases were admitted. We studied the results of drug-susceptibility of Mycobacterium to Isoniazid (H), Rifampicin (R), Ethambutol (E), Streptomycin (S), Kanamycin (K), Ofloxacin (O) in solid Lowenstein-Jensen medium. Cases of pan- sensitivity, drug resistance (DR), including MDR and extra drug resistance (XDR) were defined.

Results As much as 163 cultures were studied. From 52 cases in 2005, 36.5% were pan-sensitive, 25.0% were DR and 38.5% were MDR. In 2006, 36.0% of 50 cultures were pan-sensitive, while casualty of MDR increased to 48.0%. In 2007, number of pan-sensitive cultures decreased to 21.4%, while 35.7% were MDR and 7.2% were XDR. In 2008, among 47 cultures nearly half were MDR (48.9%) and 12.8% were XDR, only 27.7% cultures were pan-sensitive.

Conclusions Among prisoners in St. Petersburg, the value of the cases of primary MDR and XDR TB increased dramatically. Introduction of the fast methods of drug resistance detection is required.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 97 PP-26

AN INCREASE OF DRUG RESISTANCE SINCE 2001 IN MULTIDRUGRESISTANT M. tuberculosis ISOLATES FROM BELGIUM

Karolien Stoffels, Maryse Fauville-Dufaux Reference Laboratory of Tuberculosis and Mycobacteria, Scientific Institute of Public Health, 642 Rue Engeland, 1180 Brussels, Belgium. Tel. +32-23733210 | Fax. +32-23733281. [email protected], [email protected]

Between January 1994 and December 2008, MDR clinical isolates of 174 patients were analyzed in our National Reference Laboratory. They represent 90% of all the MDR-TB patients identified in Belgium during this 15-years period. Since 2000, the number of MDR patients identified in our country is stable (in average 16 per year, i.e. an average of 1,3 % of the patients tested for susceptibility to drugs) but the isolates are resistant to more and more second line drugs. We observe a dramatically increase in resistance to ethambutol, rifabutin, amikacin and ofloxacin, as well as to pyrazinamid. We divided the studied period in 2 ranges, 1994 to 2000 and 2001 to 2008. Only the first MDR clinical isolate of each patient was taken into account. So these results do not consider the evolution of the isolates during treatment in Belgium, but only the initial MDR resistance profile. In the second period (2001 to 2008) 75,6% of the MDR clinical isolates showed resistance to ethambutol versus 45,5% in the first period (increase of resistance of 30,1%); 75,4% were resistant to rifabutin versus 70,4% in the first period (increase of 5%). Resistance to pyrazinamid increased from 39,6% to 55,6% (difference of 16%). Resistance level to amikacin showed an increase of 12,2% (3,6% to 15,8%) and resistance level to ofloxacin showed an increase of 8,6% (3,6% to 12,2%). No primary XDR isolate was observed during the first period, but 5 were detected since 2001. Three MDR isolates developed into XDR what the total amount of XDR strains brought to 6 and only 2 for respectively the second and first period. Concerning the genetic families identified, 14,7% more Beijing strains were registered in the second period compared to the first period. A decrease of the members of the LAM and Haarlem family was noted (16,4% and 17,3%). In conclusion, an important increase of resistance to ethambutol, pyrazinamid, amikacin and ofloxacin is observed in MDR clinical isolates detected in Belgium. This confirms the urgent need for new anti-tuberculosis drugs.

98 ESM 2009 PP-27

Mutational analysis of genes associated with resistance to injectable second-line drugs in Mycobacterium tuberculosis clinical isolates from Lisbon, Portugal

João Perdigão1, Ana Ferreira1, Ana Malaquias1, Rita Macedo2, Laura Brum2 and Isabel Portugal.1,2 1 - Centro de Patogénese Molecular, URIA, Faculdade de Farmácia da Universidade de Lisboa 2 - Laboratório de Micobactérias, Centro de Bacteriologia, Instituto Nacional de Saúde Dr. Ricardo Jorge

Objectives Multidrug resistance (MDR) constitutes a serious problem to tuberculosis (TB) control program in Portugal. An even more serious threat is the one posed by the high rate of extensive drug-resistant TB (XDR-TB). Our laboratory has already shown that high rates of this form of TB exist in Lisbon. Given the fact that MDR-TB and XDR-TB are currently associated with a limited number of genetic clusters, mainly Lisboa family clusters, the diversity of genetic polymorphisms conferring resistance to second-line drugs is also probably limited. In this study we intended to characterize the genetic polymorphisms associated with resistanace to second-line injectable drugs and to assess the clinical isolates clonality.

Methods We have analyzed 19�� MDR-TB strains resistant to one or more second-line injectable drugs, collected from several hospital units across Lisbon Health Region during the year of 2005. All isolates were typed by Mycobacterial Interspersed Repetitive Units (MIRU-VNTR) and, screened for mutations in tlyA and rrs genes.

Results Three different mutations were identified on tlyA gene and another three at rrs gene. Overall, 9 isolates had mutations in tlyA gene and 8 isolates had mutations in rrs gene; two isolates didn’t have any mutation in either gene. The most frequent mutations found were A1401G in rrs gene (6/19) and 755InsGT in tlyA gene (6/19). We also verified that there was no overlapping of mutations from different genes. The genotyping analysis revealed that the isolates could be distributed through two different MIRU-VNTR genetic clusters: Lisboa3 and Q1. Cluster Q1 contained all clinical isolates bearing the A1401G mutation in rrs gene, while Lisboa 3 cluster contained all isolates that had the 755InsGT mutation in tlyA gene.

Conclusion We have identified several mutations that might be associated with resistance to different but related second-line drugs: kanamycin, amikacin and capreomycin. The two most prevalent mutations were associated with different genetic clusters, which suggests recent transmission and, ultimately, that XDR-TB transmission is taking place. The most prevalent mutations associated with injectable second-line drugs have therefore been defined, which opens the way for molecular detection of resistance to second-line drugs in the region.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 99 PP-28

MULTIDRUG-RESISTANT TUBERCULOSIS

Nuak, Joao; Ferreira, Danina; Carvalho, Teresa; Gomes, Maria Helena; Sarmento, Antonio Hospital S. Joao, Porto - Portugal

Introduction Multidrug-Resistant Tuberculosis (MDRTB) is caused by M.tuberculosis (MT) resistant to at least Isoniazid (INZ) and Rifampin (RIF), and can be due to unsuitable or irregular treatment.

Purpose To know the epidemiological and clinical characteristics of the disease in patients (pts) hospitalized with MDRTB in an Infectious Diseases Service.

Patients And Methods Review of clinical records of pts with MDRTB. Diagnosis was based on drug susceptibility testing.

Results Between 1996 and 2007, 16 pts had MDRTB. Eleven were male. Ages ranged between 27-40y(X=32.2±4.33). Fifteen (93.8%) had HIV infection (14 drug addicts; 1 sexual risk). One pt had professional contact with MDRTB. The disease was only pulmonary in 7(44%) pt, disseminated in 5(31%), pulmonary and extra-pulmonary in 3(19%)-meningeal, lymph nodes (LN) and urine in one each pt; another pt had only meningeal disease. Eleven (69%) pts had previous irregular antituberculosis treatment. MT was isolated in sputum in 12/16 pt (75%); CSF in 5/6 pt (83%), bronchoalveolar lavage in 2/6(33%), blood in 3/6(50%), urine in 2/6(33%), LN in 2/6(33%), gastric aspirate and feces in one each. Most pts had MT isolated in more than one sample. All MT were resistant to INZ and RIF. Thirteen out of 16 pt (81%) were also resistant to strep- tomycine, 6/12(50%) to pyrazinamide, 6/16(37%) to ethambutol, 5/12(42%) to rifabutine, 10/15(67%) to ethionamide, 4/14(28%) to kanamycin, 4/13(31%) to ofloxacilline, 3/7(43%) to PAS, 2/5(40%) to kapriomycine e 1/5(20%) to cyclocerin. In 4 pts MT was simultaneously resistant to at least three 2nd line drugs (XDRTB). Fifteen pts had medical treatment according to the drug susceptibilities testing. In two pts pneumectomy was performed. Eight pts died: One before diagnosis and 7 between 30-720 days of diagnosis. One pt survived for 6y. maintaining positive cultures of sputum despite medical and surgical therapy. Two pts were treated for 18 months with clinical and radiological improvement, without evidence of disease 1 and 5y. later. Five pts were lost to follow-up. One is on treatment with clinical and radiological improvement.

Conclusion MDRTB is a serious public health problem. HIV, drug addiction and irregular treatment were important factors for its development. Prognosis depends on early detection of drug resistance and institution of appropriate therapy. We emphasize the very high mortality.

100 ESM 2009 PP-29

CHARACTERISATION OF STREPTOMYCIN MUTATIONS IN Mycobacterium tuberculosis CLINICAL ISOLATES IN THE AREA OF BARCELONA

Griselda Tudó1, Emma Rey1, Fernando Alcaide2, Pere Coll3, Gemma Codina4, Núria Martín-Casabona4, Michel Montemayor3, Raquel Moure2, Margarita Salvadó5, Julià González-Martín 1 1 - Servei de Microbiologia, CDB. Hospital Clínic de Barcelona-IDIBAPS, Universitat de Barcelona 2 - Servei de Microbiologia, Hospital Universitari de Bellvitge-IDIBELL, Universitat de Barcelona 3 - Servei de Microbiologia, Hospital de la Santa Creu i Sant Pau de Barcelona, Universitat Autònoma de Barcelona 4 - Servei de Microbiologia, Hospital Universitari Vall d’Hebron. Universitat Autònoma de Barcelona 5 - Laboratori de Referència de Catalunya, Barcelona. All the authors are members of Spanish Network for the Research in Infectious Diseases (REIPI, RD06/0008).

Objective To determine the proportion and type of mutations in Mycobacterium tuberculosis (Mtb) isolates resistant to streptomycin (SM) and their relationship with the level of resistance and their genotype.

Methods SM resistant isolates from an Mtb strain collection (1995-2007) were studied. Minimum inhibitory concentrations (MIC) of SM for each isolate were determined using the proportion method with Middlebrook 7H10 medium. The entire rpsL gene and 2 specific fragments (loop 530 and region 912) of the rrs gene were sequenced. IS6110-RFLP and spoligotyping techniques were used to type Mtb isolates.

Results Of 69 SM resistant isolates, 36 (52.17%) presented a mutation in either the rpsL gene and/or the rrs530 gene, with no mutation in the rrs912 region. No mutations were found in 33/69 (47.8%) SM resistant isolates (all of them with MIC≤16µg/ ml). Seventeen (24.63%) isolates showed rpsL mutations: 9 (13.04%) at position 88 (7: AAG→AGG, 1: AAG→ACG and 1: AAG→CAG) and 8 (11.59%) in codon 43 (AAG→AGG). Isolates with mutations in the rpsL gene (94.1%) had a MIC≥512 µg/ml. Among isolates with alterations in the rrs gene (27.53%):10 (14.49%) had a 491 C→T change and low MIC level; 7 (10.1%) had a mutation at position 513 A→T or A→C and 2 (2.89%) had a 516 C→T substitution. These mutation points were related to intermediate and high MIC levels. One isolate with a codon 88 mutation had a second mutation in the rrs530 gene at position 491. IS6110-RFLP typing identified 4 clusters (11/69, 13%). Clusters I and II were monoresistant to SM, with a low MIC level and a mutation at position 491 in the rrs gene. Cluster III was multidrug-resistant with a high MIC level and a mutation in codon 88 in the rpsL gene. Cluster IV and V was monoresistant to SM with a low MIC level and no mutation. Interestingly, all the isolates with a mutation at position 491 in the rrs530 gene were identified as LAM3 lineage. All the Beijing family presented mutations in the rpsL gene (2 and 1 at codons 88 and 43, respectively).The spoligotyping lineageT5-MAD2 was detected in non-mutated isolates.

Conclusions Mutations in the rpsL and rrs genes were detected in at least 50% of SM resistant isolates. Mutations in the rpsL gene were associated with high-level resistance while mutations in the rrs530 gene were associated with different MIC levels. The isolates with no mutations had low-level resistance. Mutations in the rrs530 gene at position 491 were associated with LAM3 lineage

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 101 PP-30

SURVEILLANCE OF DRUG-RESISTANT TUBERCULOSIS IN ITALY

Fattorini Lanfranco 1, Pardini Manuela 1, Cirillo Daniela 2, Borroni Emanuele 2, Miotto Paolo 2, Filippini Perla 3, Cassone Antonio 3, TB-CCM Study Group 4 1 - Istituto Superiore di Sanità, Rome, Italy 2 - Istituto Scientifico San Raffaele, Milan, Italy 3 - Istituto Superiore di Sanità, Rome, Italy 4 - Italian network of mycobacteriology laboratories

Introduction Drug-resistant TB is an increasing problem worldwide. In order to update the national data on drug resistance, we started a surveillance program including the most representative diagnostic centres in Italy. Purpose of the study. The study was designed to determine: 1) Accuracy of drug susceptibility testing (DST) for streptomycin (S), isoniazid (I), rifampicin (R), ethambutol (E). 2) Drug resistance in new cases (NC), previously treated cases (PTC), patients born in Italy (PBI), patients born abroad (PBA). 3) Molecular typing.

Methods 1) Thirty laboratories were enrolled in 2006-7 in 18/20 regions for proficiency testing (PT), based on the amount of DST performed every year and geographic location. The laboratories received 20 strains each, and sent DST results to the WHO Supranational Reference Laboratory (SRL) of Istituto Superiore di Sanità (ISS) which compared them with the judicial results of the Global Network of SRLs. 2) A questionnaire was returned to ISS with DST results of TB cases diagnosed in 2007; strains resistant to >1 drugs and 10% of the susceptibles were collected. 3) molecular typing was performed at the SRL of San Raffaele Hospital (Milan) by spoligotyping and MIRU-VNTR. Results. 1) Twenty-nine laboratories completed the PT. The average efficiency (correct results/total results) was high (95.5±2.8% for S, 97.6±2.9% for I, 95.7±2.9% for R, 96.2±4.4% for E). 2) In 2007, a total of 1,698 antibiograms for SIRE were examined. As to NC and PTC, total monoresistances were 10.6 and 16.5%, respectively; MDR cases were 2.5 and 26.6%, respectively. As to PBI and PBA, total monoresistances were 10.9 and 11.2%, respectively; MDR cases were 2.6 and 5%, respectively. 3) In 257 strains collected in 2006-07 and examined for molecular typing, 146 spoligotypes and 29 clusters were found. Beijing genotype was detected in 5% of cases. In 44 MDR strains typed by the MIRU-12 technique the clustering rate was 0.023 showing low transmission rate.

Conclusions 1) PT indicated that the DST in this network of laboratories was accurate. 2) MDR rate in Italy consistently increased in NC from 1998-2001 (1.1%) to 2007 (2.5%), a phenomenon likely related to immigration. 3) No MDR-TB outbreak was detected. Noteworthy, unlike reported from other European countries, MDR strains were not associated with the Beijing lineage (This work was supported by the Italian Ministry of Health, CCM Project Surveillance of resistance to anti-TB drugs, Grant N92)

102 ESM 2009 PP-31 tuberculosis RESISTANCE in a general hospital in portugal – 9 years surveillance

Sancho L.; Portugal C.; Tancredo L.; Silva M.; Dias A.; Silva F.; Sousa Germano Laboratory of Microbiology, Department of Clinical Pathology Hospital Fernando Fonseca – Amadora, Portugal [email protected]

Tuberculosis remains a serious public health problem in Portugal. In 2008, the Portuguese Health Authorities reported a TB incidence of 25,3/100.000 inhbitants (13,6% immigrants). TB Multi Drug Resistant (MDR) were 2,5%, 34% of which were Extensively Drug Resistant (XDR). Resistance to any of the primary drugs makes the disease more difficult and expensive to treat. Our Hospital is located in Lisbon’s surroundings and covers a population of 750.000 inhabitants most of them with poor socioeconomic level and immigrants from Africa and East Countries. In the Great Lisbon are located 66% of TB cases of Portugal.

Purpose The aim of this study was to investigate the frequency of drug resistance of Mycobacterium tuberculosis Complex in a general Hospital in Amadora, Portugal, during a 9-year period (2000-2008).

Methods A total of 19.417 clinical specimens (15.159 pulmonary and 4.261 extra pulmonary), collected from 9.525 patients, were cultured for mycobateria. Molecular genetic identification of M.tuberculosis Complex and its resistance to Isoniazid and/or Rifampicin was made with the technology Genotype MTBDR plus (HAIN-Lifecience-Germany). Antimycobacterial susceptibility test to the primary drugs, Streptomycin (STR), Isoniazid (INH), Rifampicin (RIF), Ethambutol (ETB) was performed in BACTEC MGIT 960 System

Results In 19.417 cultured clinical specimens for mycobateria, 1094 (14,2%) were positive by cultural methods. 1029 were identified as M.tuberculosis Complex; 783 (76,1%) were strains without resistance, 150 (14,6%) with one resistance, 73 (7.1%) were MDR, being more than 25% XDR. The proportion of M.tuberculosis strains resistance rate to antituberculosis drugs during the 9-year period was: Isoniazid 11,1% (114), Streptomycin 20.2% (208)), Rifampicin 7.2% (74), and Ethambutol 4.7% (48); but in 2008 was: Isoniazid 4,8%, Streptomicin 17%, Rifampicin 4,1% and Etambutol 2%. On our tuberculosis population (661), in the last 6-years (2003-2008), we have compared the resistance rate related to 3 parameters: sex, age and HIV. The tuberculosis (661) and MDR (38) populations have the same incidence rate: in male (67%) and in females (33%). The age distribution in the MDR population (38) was 0% [0-15], 5% [16-25], 29% [26-35], 39% [36-45], 13% [46-55], 11% [56-65], 0% [66-75], 3% [76-100]; and in patients without resistance (623) was 3% [0-15], 13% [16-25], 30% [26-35], 20% [36-45], 14% [46-55], 8% [56-65], 7% [66-75], 5% [76-100]. The HIV parameter results were analysed on a 554 tuberculosis population. The MDR incidence rate for the HIV group (213) was 7%, and for the no HIV group (341) was 4%.

Conclusion The level of resistance in our population (MDR 7%) is significantly higher than Portugal’s average (2,5%) The Multi Drug Resistance tends to be lower in the last years. The same can be observed in each of the tested drugs. HIV infection, age and sex patient are factors that contributed to the variation of tuberculosis/MDR incidence rate. Comparing the resistance rate by sex parameter, we didn’t found differences for tuberculosis or MDR populations; they both have a bigger incidence in the male sex (67%). The major incidence of tuberculosis is among active population, between 26 and 45 years old, but, it is between 36 and 45 that we found most of the MDR strains (39% of all), mostly because of drug abuse and HIV infection in this age group. The incidence of MDR tuberculosis is clearly bigger in HIV positive (7%) than in HIV negative (4%).

Discussion In spite of our population have a level of resistance above the Portuguese average, we noted that the number of tuberculosis cases, including MDR, decreased 7,8% during this 9-year period analysed, which is comparable to 2008 national data (-7.2% in the last decade).

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 103 PP-32

DOES A MUTATION IN THE RPOB MEAN THAT THE M.tuberculosis IS RESISTANT TO RIFAMPICIN?

Yates, Malcolm; Brown, Tim; Drobniewski, Francis HPA National Mycobacterium Reference Unit

Testing for mutations in the “hot spot” region of the rpoB gene is gaining momentum for the diagnosis of rifampicin resistant strains of M.tuberculosis and as a surrogate marker for MDRTB. The use of PCR combined with hybridisation to commercial strips (Hain Lifesciences, Innolipa)has decreased time and increased ease of use so that these investigations are being performed by more and more laboratories. The strips consist of a series of overlapping probes that cover the whole region (S bands) and are all present in Wild Type isolates. There are also a series of probes (R bands) covering the commonest mutations which are linked with rifampicin resistance and with the deletion of the corresponding S band. Occasionally an S band is deleted with no R band appearing. We report these as “unidentified mutations”. The question is whether these isolates are resistant or sensitive to rifampicin. Recently we identified three patients from Sao Tome, an island off the West Coast of Africa (population 55000), with a strains of M.tuberculosis that had the same S band deleted. Sequencing data showed that the strains had the same synonymous mutation, VNTR/MIRU profiles were identical, and all strains were fully sensitive to first line drugs. On analysis of our database, 466 strains were found to have rpoB mutations, 103 (22%) of these were unidentified mutations of which 13% were sensitive to rifampicin. A further 15 isolates gave a WT result but were rifampicin resistant. All strains were sequenced to identify the mutation. Before rpoB mutations can be used as a surrogate for MDRTB the rate of mono-resistance to rifampicin in the area must be determined: e.g. in the London area 30% of rifampicin resistance is mono. Care must, therefore, be taken when reporting these rpoB mutation results to Clinicians as 1) unidentified mutations do not always correspond to rifampicin resistance and 2) rpoB mutations may not always correspond to MDRTB

104 ESM 2009 PP-33

GENOTYPIC DETECTION OF ISONIAZID AND RIFAMPIN RESISTANCE IN Mycobacterium tuberculosis CLINICAL ISOLATES

Maitane Aranzamendi Zaldumbide, Carlos Fernandez Mazarrasa , Luis Martinez-Martinez, Jesus Agüero Balbin. Hospital Universitario Marques de Valdecilla, Servicio de Microbiologia, Santander, Spain.

Background The emergence of Mycobacterium tuberculosis resistant to first-line drugs underlines the urgent need for new resistance- profiling methods that would allow for timely determination of proper treatment. The aim of this study was to evaluate the development of targeted and fast molecular diagnostic method suitable for specific genome regions responsible for isoniazid (INH) and rifampin (RAMP) resistance in M. tuberculosis clinical isolates.

Methods 79 strains known to be resistant to INH (n=71) or INH+RAMP (n=8) by the automated system BacT ALERT (bioMérieux) were selected from a stock collection of clinical isolates (January 2000- March 2009). The genome regions associated with INH-R (including the codon 315 of the katG gene and the fabG1(mabA)-inhA regulatory region) and RAMP-R (81-bp hot spot region of the rpoB gene called RRDR) were amplified by PCR and the DNA sequences were studied.

Results Of the 79 isolates, 22 (27.84%) had the mutation S315T in the katG gene, 5 (6.32%) showed changes at -15 nucleotide of the fabG-inhA regulatory region and 2 (2.53%) presented both mutations. A significant proportion of strains, 50 (63.29%), had no detectable alterations at the studied loci. INH + RAMP-R strains were associated with mutations in the RRDR of the rpoB gene in all cases. From these, majority (5 of 8, 62.5%) presented the mutation S531L, whereas the others involved changes at the codon 516: mutations D516V and D516F, which were identified in 1 (12.5%) and in 2 (25%) strains respectively.

Conclusions Our results demonstrated a low sensitivity of this method to detect INH-R strains, and points to the need of finding out other mutant regions. On the other side, confirm the usefulness of this strategy for fast assessment of resistance to RAMP, which in turn is a marker for multiresistance. This analysis can also be done with assays based on reverse line blot hybridization detecting the same mutations, except D516F, which is not targeted.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 105 PP-34

PHYSIOLOGICAL FITNESS AND TRANSMISSION POTENTIAL OF MULTI-DRUG RESISTANT Mycobacterium tuberculosis CLINICAL ISOLATES IN HONG KONG

CHAN Chiu Yeung Raphael 1, CHAN Wai Chi Edward 1, AU Tai Kong Mike 1, LAI Wai Man Raymond 2, YEW Wing Wai 3, YIP Chi Wai 4, KAM Kai Man 4. 1 - Department of Microbiology, the Chinese University of Hong Kong; 2 - Department of Microbiology, Prince of Wales Hospital 3 - Tuberculosis & Chest Unit, Grantham Hospital, 4 - Tuberculosis Reference Laboratory, Department of Health, HKSAR, Hong Kong.

This study evaluated the infectivity and transmission potential of multi-drug resistant Mycobacterium tuberculosis (MDR- MTB) strains by determining (i) whether resistance developed at a physiological cost which rendered them less capable of surviving environmental stress and infecting human host, and (ii) the degree of genetic relatedness shared by resistant organisms, which indicated the extent by which they spread among humans. The relative growth rates of selected isolates were measured using the MGIT system and compared to that of drug-sensitive strains. Our data showed that their average initial growth rate, measured within 7 days of inoculation, was inversely proportional to the number of mutations they harbored in key resistance genes, with strains carrying 5 mutations growing at a rate 46% slower than that of the wild type. These findings suggested that resistance gene mutations in MTB imposed a range of physiological cost characterized by reduced growth fitness. However, results of epidemiological typing showed that 34% of the 402 MDR-MTB isolates analyzed exhibited genetic relationship to other strains, indicating that such fitness cost did not significantly affect the ability of MDR-MTB to transmit between human hosts and cause infection. Importantly, the identification of five major clusters of 50 strains strongly suggests that infection due to dissemination of ‘parental’ MDR-MTB clones is common. On the other hand, the majority of test strains displayed a unique genetic profile, indicating that MDR-MTB might also emerge independently through drug selection within individual patient. The DNA fingerprints and growth fitness data of local resistant isolates may be used for matching the genetic identities, predicting transmission potential, and tracing the routes of dissemination of future MDR-MTB isolates in the community. This work was supported by the Research Fund for the Control of Infectious Diseases (Project code 6902191 and 6902200)

106 ESM 2009 PP-35

Mycobacterium tuberculosis: 1999-2008 ANTITUBERCULOSIS DRUGS SURVEILLANCE IN CLINICAL ISOLATES FROM PATIENTS IN THE LARGEST HOSPITAL IN THE NORTH OF PORTUGAL

Sousa, Ana Sofia; Pinheiro, Maria Dolores; Carvalho, Teresa; Gonçalves, Helena Laboratório de Microbiologia do Serviço de Patologia Clínica. Hospital de S. João, Porto, Portugal

Introduction Tuberculosis, whose causal agent is Mycobacterium tuberculosis (MT), is still an important cause of morbidity and a leading cause of death by infection worldwide. Multidrug resistant (MDR) and extreme resistant drug (XRD) strains of MT are frequently isolated. They have become an emerging global public health problem and an obstacle for tuberculosis (TB) control. Insights on prevention of disease dissemination and its empirical treatment may be obtained from resistance surveillance and monitorization of its trends. Therefore, in our study we present the susceptibility data of the strains isolated in Hospital de S. João over the past ten years.

Material and Methods A prospective ten year study was done using susceptibility data of MT strains isolated in the hospital. Susceptibility testing to first line drugs (streptomycin, isoniazid, rifampin and ethambutol) was made on the first isolate of each patient, using the proportion method (in Lowenstein-Jensen medium until 2000, from 2001 to 2003 in Bactec 460TB and in Bactec MGIT thereafter). When MDR strains were present, second line drugs (ethionamide, cycloserine, capreomycin, kanamycin, amikacin, ciprofloxacin and rifabutin) were tested.

Results In these 10 years, 1493 MT strains isolated for the first time in the same number of patients, including 1103 males, were tested for in vitro susceptibility. Streptomycin was the drug with high index of resistance (8,8%); isoniazid (6,0%) was the second one; rifampin (2,4%) the third and ethambutol (1,3%) was the less resistant. We found no resistance in 1328 (88,9%) of our patients, but in 29 (1,9%) we isolated MDR strains and 13 (0,8%) patients had XDR ones. From 1999 to 2005, the number of MDR strains isolated was relatively stable (an average of 3 patients/year); in 2006, 2007 and 2008 the number of patients with MDR was 8, 3 and 1 respectively. In what XDR strains are concerned, we had no isolates from 1999 to 2002; in the following years, 2003-2008, XDR strains were isolated in 1,0,2,7,2 and 1 patients.

Conclusion The results obtained in our patients are similar to previous reports in Portuguese and international literature, supporting the view that tuberculosis is currently a serious public health problem. The knowledge of susceptibility results will provide evidence in support of preventive health policies. It also emphasizes the role of Laboratory as a cornerstone in diagnosis, management of individual patients and effective TB control.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 107 PP-36

FITNESS COST OF Mycobacterium tuberculosis CLINICAL ISOLATES RESISTANT TO FLUOROQUINOLONES

VON GROLL, Andrea 1; MARTIN, Anandi 1; JUREEN, Pontus 2; HOFFNER, Sven 2; PORTAELS, Françoise 1; PALOMINO, Juan Carlos 1; ALMEIDA DA SILVA, Pedro 3 1 - Institute of Tropical Medicine, Antwerp, Belgium 2 - Swedish Institute for Infectious Disease Control, Solna, Sweden 3 - Universidade Federal do Rio Grande, Rio Grande, Brazil

Fluoroquinolones (FQs) have been used as effective second-line drugs in the treatment of the tuberculosis. However, the emergence of M. tuberculosis resistant to FQs has contributed for the occurrence of XDR-TB. This study investigated the fitness cost related to the mechanism of resistance to FQs in M. tuberculosis clinical isolates. A total of 37 isolates had the ofloxacin, moxifloxacin and gatifloxacin susceptibility determined by the proportion method and were sequenced to look for mutations in gyrA and gyrB. The role of efflux pumps was evaluated by determining the minimal inhibitory concentration of the FQs in the presence and absence of verapamil by resazurin microtiter assay. Growth curves of the isolates were obtained using the MGIT960 automated system and the lag phase time and rate of growth were established to compare the fitness. On the 25 FQ resistant isolates (FQR), the most frequent mutation was at Ala-90→Val, followed by mutations at Asp-94 to Gly, Tyr, Ala and Asn. Some unusual mutations were identified at Asp-89→Asn, Asn-533→Thr (gyrB) and deletion of the codon 678 and 679 in gyrB. The isolate with mutation at Asn-533→Thr was the only case of no whole cross resistance among the three FQs tested. One FQR isolate was wild type for the region investigated. The efflux mechanism was indentified in 36% of the FQR isolates, being more frequently found in moxifloxacin and gatifloxacin. In regard to the fitness parameters, the mutation at Asp-94 showed a longer lag phase while the mutation at Asn-90 had not any significant difference related to the wild type FQS. The establishment of FQR isolates without fitness cost warns for the possibility of a continue emergence of XDR-TB and highlights for a more rational use of FQs, not only for the treatment of TB, but also, for other bacteria.

108 ESM 2009 PP-37

IN VITRO ACTIVITY OF OFLOXACIN, MOXIFLOXACIN AND GATIFLOXACIN AGAINST Mycobacterium tuberculosis BY THE RESAZURIN COLORIMETRIC METHOD

VON GROLL, Andrea 1; MARTIN, Anandi 1; JUREEN, Pontus 2; HOFFNER, Sven 2; PORTAELS, Françoise 1; ALMEIDA DA SILVA, Pedro 3; PALOMINO, Juan Carlos 1 1 - Institute of Tropical Medicine, Antwerp, Belgium 2 - Swedish Institute for Infectious Disease Control, Solna, Sweden 3 - Universidade Federal do Rio Grande, Rio Grande, Brazil

The in vitro activity of ofloxacin, moxifloxacin and gatifloxacin was tested against 41 strains of Mycobacterium tuberculosis by the resazurin microtiter assay (REMA) plate and the proportion method on 7H11 agar. A critical concentration of 2.0 µg/ml for ofloxacin and 0.5 µg/ml for moxifloxacin and gatifloxacin was obtained by the proportion method on 7H11 agar. For REMA we propose a critical concentration of 2.0 µg/ml for ofloxacin and 0.25 µg/ml for moxifloxacin and gatifloxacin. Full cross-resistance among the three fluoroquinolones could not be confirmed since one strain resistant to moxifloxacin and gatifloxacin was still susceptible to ofloxacin. This finding could have important implications for the treatment of tuberculosis patients.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 109 PP-38

RAPID DETECTION OF EXTENSIVELY DRUG-RESISTANT Mycobacterium tuberculosis BY THE RESAZURIN MICROTITER ASSAY PLATE

Paasch, Fabienne; Martin, Anandi; Docx, Sven; Fissette, Kristina; Portaels, Françoise; Palomino, Juan Carlos Institute of Tropical Medicine, Antwerp, Belgium

Introduction A major concern for tuberculosis control programs is the emergence of multidrug-resistant (MDR) tuberculosis and especially extensively drug-resistant (XDR) tuberculosis. Conventional drug susceptibility testing (DST) requires 3 to 6 weeks to yield results. Therefore, there is an urgent need of new timely and accurate detection of first and second line anti-tuberculosis drug resistance.

Purpose of the study The aim of this study was to evaluate the first and second line drugs rifampicin (RIF), isoniazid (INH), ofloxacin (OFX), kanamycin (KAN), amikacin (AMK) and capreomycin (CAP) with clinical isolates of M. tuberculosis by the colorimetric resazurin microtiter assay (REMA) plate in comparison to the indirect proportion method (PM).

Method A total of 150 clinical isolates were studied. DST by PM was performed on Löwenstein Jensen for RIF and INH and on 7H11 agar for the other drugs. The minimal inhibitory concentration obtained by REMA was compared with the PM.

Results REMA results were easily determined visually after 8 days compared to 21 to 42 days by the PM. Out of 150 isolates 92 were MDR and 20 were XDR. After defining the critical concentration for each drug by the colorimetric assay, excellent results were obtained for first and second line drugs with levels of specificity and sensitivity between 93% and 100%.

Conclusion In this study drug resistance detection by REMA has shown high level of agreement with the conventional PM. Therefore, REMA could be a reliable alternative method for rapid detection of MDR and XDR M. tuberculosis.

110 ESM 2009 PP-39

DETECTION OF EMBB GENE CODON 306 MUTATIONS IN ETHAMBUTOL SUSCEPTIBLE AND RESISTANT Mycobacterium tuberculosis STRAINS.

Montoro, Ernesto 1; Yzquierdo, Sergio 1; Lemus, Dihadenys 1; Echemendia, Miguel 1; Takiff, Howard 2 1 - Institute of Tropical Medicine Pedro Kourí (IPK), Havana, Cuba 2 - Venezuelan Institute for Scientific Research (IVIC), Caracas, Venezuela

Ethambutol (EMB) is one of the first line drugs in the treatment of tuberculosis. The major mechanism of acquisition of resistance to EMB in Mycobacterium tuberculosis seems to be associated with points mutations in the embCAB operon encoding different arabinosyl transferases. In particular, amino acid replacements at embB gene codon 306 occur frequently in EMB-resistant M. tuberculosis strains. However, this alteration has been also reported in multidrug-resistant (MDR) strains susceptible to EMB. The aim of this work was to detect the most frequently mutation at codon 306 in EMB- resistant strains as well as MDR strains. For this purpose, the indirect Proportional Method (PM) on Löwenstein-Jensen was carried out as susceptibility test to isoniazid (0,2 µg/mL), streptomycin (4 µg/mL), EMB (2 µg/mL) and rifampicin (40 µg/mL) on 86 M. tuberculosis strains from the collection at the National Reference Tuberculosis Laboratory. All strains that showed resistance to EMB by PM, all MDR strains and a selection of EMB-susceptible strains were extracted the DNA to obtain a fragment of 803 bp by PCR, corresponding to embB gene codon 306. All this fragments were sequenced by automatic form using appropriate primers and they data were assembled, edited electronically, and compared with wildtype gene sequences. From 34 MDR strains, only 18 showed mutations (53%) being 8 resistant and 10 susceptible strains to EMB. The 61,5% of EMB-resistant strains showed mutations at codon 306 whereas EMB-susceptible strains no had alterations in this fragment. In conclusion, the results confirm that embB gene codon 306 is responsible of majority EMB-resistant in M. tuberculosis. All mutations were founded in MDR strains and because of this, the simple detection of changes in this fragment could be considered as multidrug-resistance marker.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 111 PP-40

TOLERANCE OF MOXIFLOXACIN IN ROUTINE CLINICAL TREATMENT OF TUBERCULOSIS

Yew, Wing Wai 1, YAN, See-Wan 1, FUNG, Siu-Leung 1, CHAU ,Chi-Hung 1, CHAN, Chiu-Yeung 2 1 - Tuberculosis and Chest Unit,Grantham Hospital, Hong Kong, CHINA 2 - Department of Microbiology. The Chinese University of Hong Koong, Hong Kong, CHINA

From Sep 07 through Feb 09, in a tertiary centre of pulmonary diseases, 65 patients [all male, age 70.0;16.6 yrs (mean;SD)] with tuberculosis (TB) were commenced on moxifloxacin treatment as part of their routine drug regimens, due to intolerance /contraindication to standard first-line anti-TB agents or drug-resistant disease. 96.9% of patients received 400mg moxifloxacin once daily. The duration of moxifloxacin treatment for all patients was 51.2;37.3 days, except one who received the drug for 330 days. 28 patients (43.1%) had completed their designated duration of moxifloxacin treatment. 9 patients had nausea, thrush and headache not necessitating drug withdrawal. 15 patients (23.1%) developed probably moxifloxacin-related adverse events requiring drug cessation: QTc prolongation (5), skin rash (3), arthralgia (2), neutropenia /thrombocytopenia (2), vomiting (1), angioedema (1) and ECG T-wave inversion (1). Time to occurrence of these events was 43.7;42.0 days. A 93-yr old patient died of stroke and myocardial infarct after 3 days of treatment. 2 other patients died of pneumonia during moxifloxacin therapy. A 82-yr old patient who developed ECG T-wave inversion had sudden death 9 days after drug cessation. Another 6 patients died of serious comorbidities, such as lung carcinoma, 1–80 days after moxifloxacin cessation. Otherwise all patients responded favourably to their anti-TB therapy, with clinical, radiographic and bacteriologic improvement. Univariate but not multivariate analysis reveals that older age (>65 yrs) might increase the risk of moxifloxacin associated adverse effects (P=0.07). Similarly, mortality is associated with the number of comorbidities (P=0.025). Thus, patient tolerance to “long-term” moxifloxacin use in treatment of TB appears reasonable. However, in older individuals with significant comorbidities, vigilance should be exercised regarding putative drug-associated events of potential severity, especially those of cardiovascular nature.

112 ESM 2009 PP-41

Characterization of gidB gene in Mycobacterium tuberculosis isolates in Lisbon Health Region: role in streptomycin resistance and epidemiological markers

João Perdigão 1, Ana Sabino 1, Catarina Milho 1, Rita Macedo 2, Laura Brum 2, Isabel Portugal 1,2 1 - Centro de Patogénese Molecular, URIA, Faculdade de Farmácia da Universidade de Lisboa, Portugal 2 - Departamento de Doenças Infecciosas, Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisboa, Portugal

Objectives Streptomycin (STP) was the first antibacillary drug introduced in the treatment of tuberculosis in 1944. With the development of further antibacillary drugs, streptomycin has become less used. Development of STP-resistance is usually explained by the acquisition of mutations in rpsL gene or in the rrs gene. Our laboratory regularly isolates STP-resistant strains without any mutation in the referred genes. Recently, mutations occurring in a rRNA methyltransferase (encoded by gidB gene) were shown to be involved in the acquisition and resistance to STP. In this study, we examined the gidB gene of STP-resistant isolates in search of mutations that may explain the acquisition and STP low-level resistance on these strains.

Methods We have analyzed by sequencing and/or endonuclease analysis the gidB gene of 57 STP-resistant clinical isolates and 30 STP-susceptible clinical isolates, recovered in 2005 and 2006 from different hospital units. The entire rpsL ORF of all isolates was amplified and screened for mutations by endonuclease and sequencing analysis. All clinical isolates were also genotyped by MIRU-VNTR.

Results The gidB gene of 19 STP-resistant isolates was sequenced and two missense mutations, A80P and F12L, were found in 5 and 1 out of 19 isolates, respectively. We have found that these gidB mutations were only present in isolates without rpsL mutations. The remaining isolates were screened by endonuclease analysis for mutations A80P and K43R in gidB and rpsL genes, respectively. Overall, mutation A80P in gidB gene was found in 10/57 STP-resistant isolates; 11/14 STP-susceptible multidrug resistant isolates; and, none of 16 pansusceptible isolates. GidB mutation A80P was also associated with MIRU- VNTR genetic cluster Q1, although an independent occurrence has been identified.

Conclusion We conclude that gidB mutations may in fact explain the high number of STP-resistant strains with no mutation in rpsL or rrs, isolated in our laboratory. These mutations probably confer STP low-level resistance that may pass undetected in regular drug susceptibity testing. The independent occurrrence suggests however, that the acquisition of such mutations present an adaptative advantage.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 113 PP-42

FLUORQUINOLONE RESISTANT Mycobacterium tuberculosis ISOLATES AND THEIR MOLECULAR CHARACTERISTICS

I Gaile 2, G Skenders 1, V Leimane 1, I Jansone 2, M Bauskenieks 2, I Pole 1, V Baumanis 2 1 - State Agency of Tuberculosis and Lung Diseases, Stopini , Riga District, Latvia, LV 2118 2 - Latvian Biomedical Research and Study Centre, University of Latvia , Ratsupites 1, Riga LV 1067, Latvia

Tuberculosis incidence decreased in Latvia from 74 per 100.000 populations in 1998 till 40.3 in 2008. However, multi drug resistance (MDR) is still high and ~10% of it is extensive drug resistance (XDR). The goal of present study is analysis of fluoro quinolone (Q) resistance and genetic characteristics of an appropriate isolates. 60 MDR cultivated and resistant to Q isolates from 2001-2007 were analysed for mutations in the gyrA gene, 25 of them using primary clinical material also. Mutations were evaluated by sequencing or using in house developed modified (Giannoni et.al.2005) reverse hybridisation method, but kanamycine (K) resistance by sequencing of rrs gene fragment. 48 isolates were confirmed as typical XDR then. Genotyping by PvuII restriction and spoligotyping was performed as well retrospective case control studies. 35 isolates (58%) contained mutations in the codone 94 (D94 to G, A or H), 14 (23%) in the 90 and 3 (5%) in the codone 91. In 8 cases (13%) mutations were not found. 8 samples (14%) contained whether two mutations or wild type sequences also. 15 K resistant isolates (of 48) contained mutation in the codone A1400G, the rest were wild type. Genotyping revealed 10 clusters with 2-6 isolates in each. 62% of isolates were of Beijing genotype, but 33% to other typical in Latvia MDR genotype – C (related to LAM). The clustering rate (47%) indicates on transmission, however, small cluster size shows, that it is more among hospitalised persons or imprisoned ones. 23 patients suffered from primary XDR TB. Treatment outcome of XDR patients is 28% cured, failures 55%, the rest defaulted or died. More profound biochemical and genetic properties of these MDR and XDR isolates ought to be studied in order to improve the cure rate. Typical genotypes mutations in the gyr gene in Q resistant isolates indicate on the necessity to search among Q line new drugs affecting other metabolic processes also.

114 ESM 2009 PP-43

DESIGN OF A RAPID METHOD OF IDENTIFICATION OF A HIGHLY TRANSMITTED STRAIN BASED ON THE LOCALIZATION OF IS6110

Sofia Samper 1, Isabel Millan 2, Ana I. Lopez-Calleja 3, Patricia Gavin 1, M. Antonia. Lezcano 4 1 - Hospital Univesitario Miguel servet / I+CS / CIBER enfermedades respiratorias 2 - Universidad de Zaragoza / Hospital Universitario Miguel Servet / I+CS / CIBER enfermedades respiratorias 3 - Hospital Universitario Miguel Servet / I+CS 4 - Hospital Universitario Miguel Servet / CIBER enfermedades respiratorias

Efficient molecular methods allowed the detection of a large and unsuspected tuberculosis outbreak, involving 85 patients in Zaragoza (Spain), caused by a strain named Mycobacterium tuberculosis Zaragoza “MTZ”, representing nearly 20% of the isolates in 2001 and being still present among the isolates from our tuberculosis population. We mapped and localized 8 of the insertion sites of IS6110 in its genome observed by RFLP. The insertion sequence 6110 besides being a very useful tool in molecular epidemiology, induces loss of gene activity either by mediating deletion events or disrupting coding sequences and regulatory domains. It also could modulate expression of neighboring genes by acting as a promoter sequence, driving or enhancing their expression. In the present work, we designed a rapid method for identifying this particular M. tuberculosis MTZ. For this purpose, different pair of primers which targeted the flanking sites of IS6110 in MTZ were designed. One hundred isolates among clinical isolates already molecular typed were chosen randomly. and were tested in these isolates. Separate PCR reactions were performed for these isolates. Among the 8 sites localized, 4 of them were previously described as preferential locus for IS6110 transposition. After testing with different locations finally one intragenic insertion in Rv2823c was selected for rapid diagnosis. Only 4 of the 100 hundred samples tested were positives, being the four positives isolates MTZ. None more of the isolates were positive, the other 96 showed the same size of the amplified product than the positive control used H37Rv, indicating that IS6110 was not present.

Conclusion the mapping of the IS6110 insertion sites in the genome of “MTZ” strain resulted in both, offers clues for better understanding of the adaptability and virulence of M. tuberculosis, and the design of a rapid method for identifying this particular M. tuberculosis MTZ. Presentations Type: Poster

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 115 PP-44

DRIVING FORCES ON THE EVOLUTION OF THE PROGENITOR OF M. tuberculosis

M. C. Gutierrez 1,2, R. Brosch2, M. Marceau1, J. Tap 2, E. Bourdon2, S. Brisse2, S. Mangenot3, G. Salvignol3, V. Barbe3, C. Médigue3, and P. Supply1 1 - Institut Pasteur de Lille –INSERM 2 - Institut Pasteur, Paris 3 - CEA/DSV/IG/Génoscope, Evry, FRANCE

Genomic and functional plasticity of agents of tuberculosis (TB) suggest that they are the legacy of extremely long evolutionary fight for survival which started with their environmental ancestors that evolved to the exclusively human intracellular parasite of our days. Almost certainly, TB has impacted on humankind through pre-history. Mycobacterium tuberculosis and its earlier relatives have probably been co-evolving with Homo sapiens and its earlier relatives for hundred of thousand of years. Despite extensive research, the cause of M. tuberculosis speciation and the factors that have led to its predominance as a human pathogen are still unknown. Previous studies of rare human TB clinical isolates from East-Africa (Van Soolingen et al., Int J Syst Bacteriol 1997; Fabre et al., J Clin Microbiol, 2004; Gutierrez et al., PLoS Pathogens 2005) showed that they share many properties of other agents of TB, but radically differ in terms of a more diversified population structure and obvious traces of intra-species horizontal gene transfer (HGT). These features suggest that these smooth TB bacilli are extant representatives of a much broader and older progenitor species, named “M. prototuberculosis”. We performed c��omparative genomics on a comprehensive collection of 56 strains of “M. prototuberculosis” to understand the roles played by various evolutionary processes in shaping the structure of M. tuberculosis genome and of its population. Multi-locus sequence typing of 16 house-keeping genes and three intein-encoding sequences confirmed the large genetic diversity of the TB bacilli and suggests that the M. tuberculosis complex (MTBC) is just a particularly suc- cesful clonal lineage that has emerged from the M. prototuberculosis progenitor pool, probably involving multiple HGT episodes (clonal epidemic structure). Preliminary analysis of whole-genome sequences from the four most genetically distant strains of smooth TB bacilli indicate extensive chromosomal rearrangements and the existence of multiple genomic islands compared to MTBC genomes. Genome downsizing, mutations, HGT of genomic islands, and intra-species recombination appear as major driving forces on the evolution of the ancestor of extant M. tuberculosis.

116 ESM 2009 PP-45

Resistance, MDR and XDR of M. tuberculosis in Spain in the last years.

P. Ruiz, M. Causse, F.J. Zerolo, J. Gutierrez, M. Casal Mycobacteria Reference Center. Department of Microbiology. Faculty of Medicine. HospitL “Reina Sofía” . Córdoba. Spain.

Tuberculosis is among the leading causes of death worldwide. The World Health Organization (WHO) estimates that 32% of the world population is infected with Mycobacterium tuberculosis . The resistance to antituberculous drugs is a big problem to the control of the illness . The emergence of multidrug-resistant strains (MDR), extensively drug-resistant (XDR) and extreme drug-resistant (XXDR) strains, is a global problem that has made a considerable alarm. There is an increasing demand to determinate in vitro susceptibilities of clinical isolates to antimicrobial agents other than those considered primary drugs. The purpose of this study, was determinate the resistance, MDR and XDR strains in our Reference Center in the four last years.

Material and method We are studied 624 samples from patients suspects of tuberculosis. 355 strains of M. tuberculosis were isolates , in BACTEC MGIT 960 and Lowenstein -Jensen medium and identified using Accuprobe and GENOTYPE MYCOBACTERIA. The susceptibility testing was made for primary drugs and: Amikacin (AK) 1.0 µg/ml; Kanamycin (K) 1 µg/ml; Capreomycin (CM) 2.5 µg/ml; Ethionamyde (ETH) 5.0 µg/ml; Ofloxacin (OF) 2.0 µg/ml; Ciprofloxacin (CI) 2 µg/ml ; Moxifloxacin (MX) 2 µg/ml; Levofloxacin (LE) 4µg/ml; Rifabutin (Rb) 0.5 µg/ml ;Rifapentine 5 µg/ml and Linezolid (Lz) 1.0 µg/ml. The Bactec MGIT technique with standart protocol was strictly followed as recommended,

Results From 624 samples, in 355 were isolated M. tuberculosis and 55 (15,49 %) of theme were resistant to some of the antimicrobial agents studied. The resistance to streptomycin was 3´38 %, to rifampin 7,88%. ethambutol 1,12 %, isoniazid 11.26 % and pyrazinamide 1,97%. A 7,6 % of strains were resistant to some of the second line drugs.The MDR was 5,6 % and the XDR 1,4 %. No XXDR were isolated.

Conclusion The resistance to secondary drugs made necessary the in vitro studies. This method, BACTEC MGIT 960 is a reliable and rapid method to determinate the secondary drugs.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 117 PP-46

DETECTION OF NON TUBERCULOUS MYCOBACTERIA IN SURFACE WATERS: COMPARISON OF CULTURE METHODS

Radomski, Nicolas 1, Lucas, Francoise 1, Cambau, Emmanuelle 2, Moulin, Laurent 3, Haenn, Sophie 3, Régis, Moilleron 4 1 - Leesu, Universite Paris-Est AgroParisTech 2 - CNRMYC, CHU Saint-Louis de Paris 3 - Crecep, Etude biologie 4 - Leesu, Universite Paris-Est AgroParisTech

Since there is no evidence for person-to-person transmission, environment is considered a likely source of non tuberculous mycobacteria (NTM) infections. Particularly, environmental water, from river, lake, pond or hot spring seems being a major source of NTM. NTM has been also isolated from wastewater, from sources of drinking water, from drinking water distribution system, from tap water, and even from bottled mineral water. Among human infections caused by NTM from water origin, pulmonary infections and cutaneous infections are often described. These NTM human infections linked to water could stem from changes in water use, from human vulnerability or from increase of virulence level among environmental strains. Also, it seems necessary to determine the origin of these NTM in the environment, in order to evaluate the importance of non-clinical habitats and to detect the emergence of virulence. Lack of knowledge about life cycle of harmful bacteria from surface water, particularly NTM, require more analytical tools which are not currently standardized or adapted to environmental samples. The aim of this study is to propose an improved culture method that could be used for counting and isolating NTM from surface water and wastewater. Based on literature, we selected different bacteriological methods from medical applications that had previously been applied to water samples. Samples from the river Seine (Paris, France) were used to select a culture method that will prevent the growth of interfering microbiota, with minimal inhibition of mycobacteria. The effect of antibiotics (polymyxin B, amphotericin B, nalidixic acid, triméthoprime, azlocillin, vancomycin) and chemical decontamination process (methods using acids, bases, detergent or cetylpyridininium chloride) on have been compared. Results will be discussed and a method adapted to water with high densities of interfering bacteria will be proposed.

118 ESM 2009 PP-47

TYPING OF Mycobacterium avium subsp. avium FROM DIFFERENT SOURCES USING PVUII–PSTI–IS901 RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) IN CROATIA

Spicic, Silvio 1, Cvetnic, Zeljko 1, Pate, Mateja 2, Duvnjak, Sanja 1, Zdelar-Tuk, Maja 1, Racic, Ivana 1 1 - Croatian Veterinary Institut, Zagreb 2 - Veterinary Faculty Ljubljana, Ljubljana, Slovenia

A total of 9 M. avium subsp. avium strains isolated from bovines (N=1), pigs (N=2), wild boars (N=2) and poultry (N=4) were genotyped by PvuII–PstI–IS901 restriction fragment length polymorphism (RFLP) analysis. The isolates were collected in the period from 2001 to 2006. Revealed profiles were designated according to the nomenclature established and used in the OIE reference laboratory for avian tuberculosis in Brno, Czech Republic. Digestion with restriction endonuclease PvuII resulted in 3 PvuII RFLP profiles (F, Q and M), comprising 8-9 bands. Digestion with restriction endonuclease PstI was successfully accomplished in 8 isolates demonstrating 4 different profiles of 11-13 bands. Among them, 3 were found to be new in the database (A31, A32 and A33). Combination of PvuII–PstI digestion revealed 4 RFLP profiles (A29/F, A31/F, A32/F, A33/M). No epizootiological connection was found between the isolates expressing the predominant profile (A29/F), found in pigs, wild boars and poultry. This is the first research in the field of genotyping M. avium subsp. avium strains isolated from different animal species in Croatia.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 119 PP-48

TUBERCULOSIS IN PETS AND WILD ANIMALS LIVING IN URBAN ENVIRONMENT

Spicic, Silvio; Duvnjak, Sanja; Obrovac, Mihaela; Zdelar-Tuk, Maja; Katalinic-Jankovic, Vera; Racic, Ivana; Cvetnic, Zeljko Croatian Veterinary Institut Zagreb

Apart from pets and domestic animals humans are often in direct or indirect contact with wild animals, especially those living in Zoos. Because of their origin these animals are a possible source of infection with mycobacteria ati- pical for certain regions. In that manner, in 2004. M. africanum type I was isolated from organs of a hyrax (Procavia capensis) that died in zoological garden in Zagreb. The hyrax had been imported from United Arab Emirates (UAE). Also, in the same year, Mycobacterium tuberculosis infection was diagnosed in a dog. This was a pet who lived it’s intire life in a city (Zagreb, Croatia) and whose owner was negative on tuberculosis. Both isolates were MIRU typed on 12 locuses (2, 4, 10, 16, 20, 23, 24, 26, 27, 31, 39 i 40). Sinse no extensive researsch was done in the past with regards to molecular typisation of M. africanum with MIRU typing, we compared our results to the ones available online (data base www. miru-vntrplus. org )(ALLIX-BÉGUEC et. al., 2008). M. africanum type I isolated from the hy-

rax had a unique code (235424253422). MIRU type of M. tuberculosis isolated from the dog was identical to 8 hu-

^ ^ man isolates originating from all over Croatia in the period from 2004.-2006. Out of these 8 ^cases, 3 originated from Zagreb and County of Zagreb; 3 from neighbouring counties (Sisac ko – moslava c ka, Karlovac ka and Varaždinska) and 2 from more distant counties (Med imurska and Primorsko – Goranska). Considering that the highest tuberculosis incidence of the same MIRU genotype was localised in a 50 km radius in as many as 6 human cases we deducted that it’s very likely that these people were source of infection for this dog. According to these examples, pets and zoo animals are important links in the chain of import and spread of pre-existing mycobacteria species in urban environment.

120 ESM 2009 PP-49

IS1245-RFLP Based Genetic Relatedness Of The Mycobacterium avium subsp. hominissuis STRAINS ISOLATED FROM HUMANS, ANIMALS AND ENVIRONMENT IN CROATIA

Spicic, Silvio 1, Cvetnic, Zeljko 1, Pate, Mateja 2, Katalinic-Jankovic, Vera 1, Duvnjak, Sanja 1, Ocepek, Matjaz 2, Zdelar-Tuk, Maja 1, Krt, Brane 2 1 - Croatian Veterinary Institut, Zagreb, Croatia 3 - Veterinary Faculty Ljubljana, Ljubljana, Slovenia

Significance of infections with Mycobacterium avium, in particular subspecies M. a. hominissuis, in animals and humans is constantly increasing. Susceptibility of humans, cattle and swine to various mycobacteria and the prevalence of these bacteria in the environment, although very important from the aspect of epizootiology and epidemiology, have not been sufficiently investigated in Croatia. This study is based on massive tuberculin skin tests of cattle and swine from large farms, bacteriology and molecular identification of M. a.hominissuis isolated from domestic and wild animals, humans and environment. Genotyping of the 23 isolates was conducted by IS1245 RFLP method. The selected cut-off value for cluster designation was similarity level of 75%. A total of 5 clusters containing 86.9 % of all genotyped strains were identified. High similarity level of the profiles within a cluster was found for the isolates from man, swine from intensive breeding, deer, wild boar and sawdust from various counties. As no epizootiological links between animals and humans were identified, humans and animals could be considered as dead-end hosts. According to our results, the source of infection for humans and animals is most probably the environment.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 121 PP-50

DEVELOPMENT OF REAL-TIME PCR ASSAY FOR QUANTIFICATION OF MYCOBACTERIA IN SURFACE WATERS

Lucas, Francoise 1, Radomski, Nicolas 1, Cambau, Emmanuelle 2, Moulin, Laurent 3, Haenn, Sophie 3, Moilleron, Regis 1 1 - Leesu, University Paris-Est 2 - CNRMYC, CHU Saint-Louis de Paris 3 - Etude Biologie, Crecep

Most non-tuberculous mycobacteria (NTM) are saprophytes living in natural environments. However, some species are opportunistic pathogens involved in various human diseases. An increase in incidence of mycobacteriosis has been recognized worldwide, probably linked to changes in water use, population vulnerability. One important question aris- ing from the increasing occurrence of NTM diseases is the origin of these pathogens. Several cases showed that water play a significant role in the transmission of NTM. Indeed NTM can be found in various aquatic ecosystems, and also in water distribution systems. However their number and diversity in environmental water bodies and in wastewaters are often underscored by the actual detection methods and no standard protocol exist. Cultural studies are time consuming and poorly specific for NTM growth. Few quantitative PCR have been developed for NTM species. However, these PCR methods have only been applied to clinical samples and may not be adapted for environmental samples. The aim of this study is to develop a real-time PCR assay to quantify NTM in surface water samples. First the specificity and sensitivity towards NTM of several published target genes, such as 16S rRNA, rpoB, hsp65, ITS and gyrA and gyrB have been evaluated using the algorithm BLAST. This first screening resulted in the selection of 8 primer pairs, which specificity and sensitivity were empirically evaluated by DNA amplification of 50 microbial isolates from the river Seine (France), which belong to Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes and Mycetes. This strain library was completed with 25 NTM and other 8 Actinobacteria strains from national collections. This second step of screening resulted in the selection of two highly specific primer pairs targeting gyrB and rrs genes, showing respectively 94,83% et 93,10 % of specificity. The efficiency of the real time PCR was evaluated for both primer pairs using the strain libraries.

122 ESM 2009 PP-51

Nontuberculous Mycobacteria, isolated from patients with lung disease, from Lisboa e Vale do Tejo region, during 2008

António Amorim1*, Rita Macedo, Edna Pereira Laboratório de Saúde Pública - Micobacteriologia/Tuberculose, Administração Regional de Saúde de Lisboa e Vale do Tejo, I.P., Lisboa, Portugal *Presenting author. Phone: 00351213602520. E-mail address: [email protected]

Despite the clinical relevance of most nontuberculous mycobacteria (NMT) in pulmonary infection in immunocompetent patients is still unclear, this mycobacteria play an increasing significant pathogenic role in HIV-positive, and other immunocompromised patients. Nevertheless, no data about NTM species isolated from patients with lung disease, from Lisboa e Vale do Tejo region, are published or available until now. We carried out a study during the entire year of 2008 with the purpose of identify, determine the incidence of each specie, and correlate this data with some epidemiological data in patients with lung disease from Lisboa e Vale do Tejo region. A total of 46 patients with lung disease were detected with NTM infection. ��Among this patients the isolated nontuberculous mycobacteria were M. intracellulare (n=7, 15,2%), M. fortuitum (n=6, 13%), M. kansassi (n=6, 13%), M. chelonae (n=4, 8,7%), M. gordonae (n=4, 8,7%), M. avium (n=3, 6,6%), M. peregrinum (n=3, 6,6%), M. spp (n=3, 6,6%), M. abscessus (n=2, 4,3%), M. mucogenicum (n=2, 4,3%), M. szulgai (n=2, 4,3%), M. triplex (n=2, 4,3%), M. lentiflavum (n=1, 2,2%) and M. simiae (n=1, 2,2%). The patients with lung disease that we identified as infected with NMT have a median age of 51,6 years, 45,7% (21/46) were male and 54,3% (25/46)were female. Despite 43,5% (20/46) of our patients were HIV-state unknown, in the group of known HIV-state, 88,5% (23/26) were HIV-negative, and only 11,5% (3/26) were HIV-positive. Our results suggest that the pattern of NTM pulmonary infection, in Lisboa e Vale do Tejo, has no significant differences from those reported in the rest of Europe and USA, and also, no significant correlation with HIV status. We carried out the study during only one year, so, further studies are needed to better clarify the NMT infection in patients with lung disease, in Portugal, in both immunocompromised and immunocompetent patients.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 123 PP-52

NONTUBERCULOUS MYCOBACTERIA INFECTIONS IN THE STATE OF PARÁ, AMAZON REGION, BRAZIL

Lima, Karla Valéria Batista 1, Lopes, Maria Luíza 1, Furlaneto, Ismari Perini 2, Lima, Elys Juliane Cardoso 2, Conceição, Emilyn Costa 2, Sousa, Maísa Silva de 2, Costa, Ana Roberta Fusco 1 1 - Instituto Evandro Chagas, Belém, Pará, Brazil 2 - Universidade Federal do Pará, Belém, Pará, Brazil

Introduction The genus Mycobacterium currently has more than 130 species. This genus includes M. tuberculosis complex and M. leprae and other organisms referred to as nontuberculous mycobacteria (NTM). In recent years, there has been a marked increase in the number of cases of human disease due NTM, and the NTM diseases seems to be related to the geographic distribution of these species in the environment.

Purpose of the study The aim of the present study was to describe the diversity of NTM from clinical isolates received at the Instituto Evandro Chagas, Pará, Amazon Region of Brazil, between 2004 and 2008.

Methods NTM included in this study were isolated from clinical specimens of 95 patients, whom 84 had pulmonary infection, and 11 infections cases related to other sites (lymphonod, biopsy and abscess fluid). Löwenstein-Jensen medium was used for the recovery of mycobacteria from clinical specimens. Genetic characterization to species level was determined by PCR-RFLP analysis of hsp65 gene (PRA), and 16S rDNA and hsp65 sequencing.

Results Ninety five patients presented NTM infections, of whom 88.4% (84/95) manifested pulmonary symptoms, 1.1% (1/95) presented lymphadenopathy and 10.5% (10/95) represented cases of healthcare-associated infections. A total of 13 species were identified and included: M. abscessus; M. bolletii; M. massiliense; M. fortuitum; M. avium; M. intracellulare; M. scrofulaceum; M. colombiense; M. kansasii; M. simiae; M. interjectum;M. smegmatis and M. szulgai. M. chelonae-M. abscessus (26), M. avium-M. intracelullare-M. scrofulaceum (27) and M. simiae (23) complexes were the most frequent in pulmonary infections. Lymphadenopathy case was caused by M. fortuitum infection. We encountered M. chelonae-M. abscessus complex (6), M. smegmatis (2) and M. fortuitum (2) in cases of healthcare-associated infections.

Conclusion We showed the diversity of species associates the NTM infections isolated at the Instituto Evandro Chagas and we encountered high variety of species in isolated from pulmonary samples. A finding was the presence of M. simiae complex species in human infections, which is not common.

124 ESM 2009 PP-53

EVALUATION OF HSP65, TB ,SP REGIONS IN IDENTIFYING MYCOBACTERIUM OTHER THANTUBERCULOSIS( MOTT);USING PCR-RFLP

Noorolhoda Saadaee Jahromi ,Shima Seif, Parissa Farnia, Mehdi Kazempour,Mohammad Kargar, JamilehNowroozi, Mehdi Kazempour, Mohammadreza Masjedi,Aliakbar Velayati Mycobacteriology Research Center (MRC),National Research Institute Of Tuberculosis and Lung Disease(NRITLD),Shahid Beheshti University Medical Campus.Tehran,Iran.

Background Mycobacteria Other Than Tuberculosis (MOTT) are frequent causes of pulmonary infections resembling TB, but differ from MTB complex by being opportunistic pathogens and are acquired mainly from the environment. Recent investiga- tors reported an increasing cause of pulmonary infection by MOTT species, in Iran. Identification of MOTT by conventional biochemical methods is cumbersome and time-consuming. Therefore in the present study,the capabilities of 3 different regions (Tb,Sp,Hsp65) were examined using three primers. We demonstrated that Hsp 65 genotyping would be very helpful to identify mycobacteria at the species level.

Material & Method DNA were extracted from 121 culture positive specimens during the year 2007-2009.The amplification carried out using following primers ( Tb ; Tb 115’-ACCAACGATGGTGTGTCCAT-3’ ,Tb 12 5’-CTTGTCGAACCGCATACCCT) ,(Sp

; Sp 15’-ACCTCCTTTCTAAGGAGCACC-3’ ,Sp 2 5’- GATGCTCGCAACCACTATCCA-3’), ( Hsp ; HSP F3 5’-ATCGC-

CAAGGAGATCGAGCT-3’ , HSP R4 5’-AAGGTGCCGCGGATCTTGTT-3’) The PCR products (Tb: 439bp ,Sp: 250-330 bp ,HSP: 644 bp) were digested using restriction enzymes with BsteII and HaeIII for Tb ,HaeIII for Sp and AvaII ,HphI,HpaII for HSP regions.The digested patterns were analyzed on 2% agarose gel.

Result The results demonstrated different sensitivity ratio for various Mycobacterium species by Tb ,Sp and Hsp regions. For RGM , Rapid Growing Mycobacteria , group(e.g., M. furtitium and M. chelonei ) the sensitivity of Tb primer was the highest among the other two regions(92%). Although, for slow growing mycobacterium(nonphotochromgen & phtochromgen) the combination of two primers(i.e., Tb+Sp) was required . Thereafter ,the sensitivity for identification of such species reached upto 94%. Incontrast , scotochromoghen(e.g M. gordonae and M. scrofalceum) were differentiated more precisely using Sp primer(74.3% ).

Conclusion The recent increase in MOTT species within the country , underline the need to rapidly distinguish such Mycobacteria from tuberculosis complex .We demonstrate the use of combined primers for accurate identification of mycobacterium up to species level.

Key Word: Identification,Atypic Mycobacteria,PCR-RFLP,RGM

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 125 PP-54

Mycobacterium avium alveolitis after cleansing hotel spa whirlpools

Svensson; Erik 1; Ridell; Malin 1; Åkerström; Magnus 2; Andersson; Eva 2 1 - Institute for Biomedicine, University of Gothenburg 2 - Department of Occupational and Environmental Medicine, University of Gothenburg

Hotel staff cleaning spa whirlpools and filters became ill in a disease suspected to be the so-called hot tub lung, which is an allergic alveolitis-like granulomatous lung disease. In total seven employees at three hotels were involved. Mycobacterium sp. was suspected to be the cause. Cultures from patients and from water and outlet filters were done. A quantitative culture method was developed and water from different parts of the equipment was analysed. One patient had definite allergic alveolitis and M. avium was isolated. Two other employees from the same hotel had suspected alveolitis, but no cultivation for mycobacteria was done. In this hotel the cleansing of the spa filters was done with high-pressure washers. Two employees at another hotel had fever, chills and dyspnea related to cleansing the equipment. Their disease was not regarded as allergic alveolitis, but both of them were colonized with M. avium In the third hotel, two employees were colonized with M. avium, but no one hade symptoms related to work. One patient, though, had flu like symptoms shortly after bathing in the pool. In respiratory samples from five of the seven patients M. avium was isolated. The pool water and the surface films of the water filters contained M. avium, often mixed with other, rapidly growing mycobacteria, however a pure culture was never obtained. In the quantitative water culture 0 - 16000 CFU/mL of M. avium was isolated. These are the first reported cases of hot tub lung alveolitis (hypersensitivity pneumonitis) in Sweden. Cultures from patients, filters and water have contained M. avium. The symptoms of the patients have presently ceased. The cleaning practices have been changed and the pool filter equipment has been rebuilt.

126 ESM 2009 PP-55

IDENTIFICATION OF NONTUBERCULOUS MYCOBACTERIA IN CLINICAL SAMPLES USING MOLECULAR METHODS: A THREE-YEAR STUDY

Isabel Couto1,2*, Diana Machado1, Miguel Viveiros1,3, Liliana Rodrigues1,4 and Leonard Amaral1,3,4 1 - Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Lisbon, Portugal 2 - Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e Tecnologia, UNL, Caparica, Portugal 3 - COST ACTION BM0701 (ATENS) 4 - UPMM, IHMT/UNL, Lisbon, Portugal

Although Mycobacterium tuberculosis, the etiologic agent of human tuberculosis is the main cause of mycobacteriosis in Man, other species of mycobacteria may also cause infection in humans. The increasing importance of nontuberculous mycobacteria (NTM) is now consensually recognized and demands for faster methods for their identification and selection of appropriate therapy. In this work we report our experience on the identification of NTM received from 12 hospitals of the Lisbon Health Region (Portugal) over a three-year period using the GenoType Mycobacterium (CM/AS) assays (HAIN Lifescience). From 1 January 2005 to 31 December 2007, our laboratory received a total of 1192 acid-fast bacilli (AFB) positive isolates from 1174 patients presenting with presumptive active mycobacteriosis. All isolates were processed for Ziehl-Neelsen staining and inoculated into MGIT tubes of the BACTEC MGIT 960 system. M. tuberculosis isolated from culture were identified by the Accuprobe system (Gen-Probe). Full-grown AFB cultures, negative for M. tuberculosis, were identified by GenoType Mycobacterium (CM/AS) kits. Out of the 1192 specimen received, 1181 were identified as members of the Mycobacterium genus. From these, 1032 cultures (87.4%) were positive for M. tuberculosis complex. The remaining 149 cultures were NTM, corresponding to 12.6% of the total number of cultures from which mycobacteria were isolated. During the study period, NTM prevalence increased steadily, starting with 8.7% in 2005 and rising to 15.2% in 2007. The joint use of the CM and AS kits identified 96.6% of all NTM isolates tested. Among the 18 NTM species identified, M. avium complex was the most frequent, although it accounted for only 34% of all NTM. In countries with high incidence of tuberculosis and, particularly, multidrug resistant tuberculosis (MDRTB) such as Portugal, therapeutic failure with isoniazid and rifampicin is anticipated to be due to an MDRTB strain. Since many NTM species are resistant to these drugs, the identification of the mycobacteria causing therapeutic failure (MDRTB versus NTM) is of major importance. The introduction of molecular methods for the identification of NTM in our laboratory has resulted in an increased awareness of the importance of being able to rapidly identify NTM as potential pathogens and the key role played by the laboratory in assisting the selection of therapeutic modality.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 127 PP-56

MYCOBACTERIA IN ANIMALS IN SLOVENIA –

AN OVERVIEW OF THE LAST DECADE ^

1 1 2 1 Mateja PATE , Darja FERME , Manca ŽOLNIR^ DOVC , Matjaž OCEPEK 1 - Veterinary Faculty Ljubljana, Gerbiceva 60, SI-1115 Ljubljana, Slovenia; e-mail: [email protected] ; fax: +386 1 4779 352 2 - University Clinic of Respiratory and Allergic Diseases Golnik, Golnik 36, SI-4204 Golnik, Slovenia

Successful national control program carried out between 1962 and 1973 contributed to eradication of bovine tuberculosis (BTB) in Slovenia. Since then, infections with the causative agents of BTB were seldom detected. The main role of veterinary mycobacteriologists has therefore become the detection and identification of the causative agents of avian tuberculosis and opportunistic mycobacterial pathogens. The aim of this study was to summarize the work done in the past ten years (1999-2008) in the field of veterinary mycobacteriology in Slovenia. Identification of the isolates was based on the following features and tests: colony morphology and growth characteristics, biochemistry, PCR and commercial identification kits AccuProbe (Gen-Probe) and GenoType (Hain Lifescience). From 1999 to 2008, a total of 292 mycobacteria were isolated from domestic, pet and wild animals in captivity. The vast majority of isolates were represented by M. avium (80.1%), identified to subspecies level in 92.7% (M. a. subsp. avium – 36.3%, M. a. subsp. hominissuis – 56.4%), while 7.3% isolates remained identified as M. avium. These mycobacteria were found predominantly in pigs, followed by cattle, poultry and exotic birds. M. caprae was found in 1.4% cases and was related to an outbreak in a zoo, affecting bisons and camels, and to a single culture-positive case of BTB in cattle in the last 15 years. M. tuberculosis was found in one cow (0.3%) as a consequence of human-to-animal transmission proven by means of molecular epidemiology. Other species detected included M. terrae (0.7%, pigs), M. fortuitum (0.7%, pig & cattle), M. scrofulaceum (0.3%, bison) and M. celatum (0.3%, pig). A relatively large proportion of isolates (16.1%) originating from pigs, cattle, sheep, goat, mouflon, bison and monitor lizard were identified to the genus level only. This could be partly attributed to the lack of reliable identification methods in the past. Development of diagnostic kits based on molecular tests led to improved and easier diagnostics of mycobacteria, especially of the species commonly found in the environment, therefore reducing the burden of unidentified mycobacteria in a routine laboratory.

128 ESM 2009 PP-57

ISOLATION AND IDENTIFICATION OF Rhodococcus AND Nocardia GENDERS IN SPUTUM SAMPLES WITH TUBERCULOSIS SUSPECT

Leite, Sérgio Roberto A; Silva, Paulo; Sato, Daisy Nakamura; Santos, Adolfo; Carlos Barreto; Miyata, Marcelo; Leite, Clarice Queico Fujimura São Paulo State University

Species from Rhodococcus and Nocardia genders are partially alcohol acid resistant and could be isolated from clinical samples (sputum, bronchial-alveolar rinsing and lung biopsy). These genders grow successfully in Löwenstein-Jensen medium and can cause pulmonary infections similar to tuberculosis cases. Our purpose was to evaluate the presence of these bacteria from 1636 sputum samples of patients with clinic suspect of pulmonary tuberculosis at Ribeirão Preto city, São Paulo state, Brazil, between the years 2000 and 2002. The samples were analyzed in the laboratory of Instituto Adolfo Lutz, Ribeirão Preto unit. From 426 acid-fast bacilli positive sputum samples, applying phenotypic methods and chemotax- onomy, we isolated 296 mycobacteria (identified as 224 M. tuberculosis and 72 NTM), 29 Nocardia sp., 51 Rhodococcus equi and 09 non-identified partially acid-fast bacilli (PAFB). The success of the treatment depends on early diagnosis, thus the differential diagnostic between Mycobacterium, Nocardia and Rhodococcus genders deserves special importance. Due to the high incidence of tuberculosis and the similarity in symptoms, probably the nocardiosis and rodococosis are under-notified in Brazil. Therefore human health professionals need to observe carefully the importance of Rhodococcus equi and/or Nocardia sp., that were found on 12% and 6.8% respectively of patients suspected with tuberculosis, attended at Ribeirão Preto city between 2000 to 2002.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 129 PP-58

PCR-RFLP OF hsp65 FOR IDENTIFICATION OF Mycobacterium leprae DIRECTLY FROM A CLINICAL SAMPLE

Neonakis Ioannis K1, Kontos Fanourios2, Gitti Zoe1, Baritaki Stavroula1, Bazigos Stavros1, Mihailelis Efstratios1, Zerva Loukia2, Spandidos Demetrios A1 1 - Microbiology Labolatory, University Hospital of Heraklion, Heraklion, Greece. 2 - Clinical Microbiology Laboratory, Medical School of Athens, “Attikon” University Hospital, Athens, Greece.

Introduction Mycobacterium leprae cannot be cultured in vitro. The application of PCR-RFLP analysis of hsp65 for identification of M. leprae had been previously proposed (Rastogi et al., J Clin Microbiol, 1999, 37, 2016-19) and, to our knowledge, the method has been used only once.

Objective The identification of M. leprae directly from a clinical sample by application of PCR- Restriction Fragment Length Polymorphism analysis (PCR-RFLP) of hsp65 gene.

Materials and Methods A sample, taken with a swab from open lesions with exudates from a 51-y old patient suspected of suffering from leprosy, was suspended in sterile water. Mycobacteria were heat-inactivated at 80o C for 1 h and the DNA was extracted using the guanidinium thiocyanate lysis buffer (Casas et al., J Med Virol, 1995, 47, 378-385). PCR amplification of a 439-bp fragment of hsp65 was performed using the protocol and primers Tb11 and Tb12 as previously described (Telenti et al., J Clin Microbiol ,1993, 31, 175-178). The PCR product was further analyzed by sequencing and RFLP. The amplified PCR product was digested using the Hae III and BsteII (New England Biolabs) restriction enzymes and the mixtures were electrophoresed on a 3% Metaphor agarose.

Results The BstEII digestion produced two fragments of 315 and 135 bp and the HaeIII digestion produced two fragments of 265 and 130 bp. This profile matched the one previously reported for M. leprae. Sequencing of the PCR product (GenBank accession number: FJ497239) verified the identity of M. leprae.

Conclusions PCR-RFLP could be a useful molecular tool as an adjunct to careful clinical and pathological assessment of patients suspected of suffering from leprosy.

130 ESM 2009 PP-59

A CASE-REPORT OF Mycobacterium thermoresistibile FROM GREECE.

Neonakis Ioannis K1, Kontos Fanourios2, Gitti Zoe1, Baritaki Stavroula1, Kosmadakis Georgios1, Baritaki Maria1, Zerva Loukia2, Spandidos Demetrios A1 1 - Microbiology Laboratory, University Hospital of Heraklion, Heraklion, Greece. 2 - Clinical Microbiology Laboratory, Medical School of Athens, “Attikon” University Hospital, Athens, Greece.

Mycobacterium thermoresistibile is a non-tuberculous mycobacterium that was first recovered in Japan by Tsukamura in 1966. Although it is strongly associated with pulmonary and dermal diseases, there have only been six reports of its isolation from clinical samples. Here we report on the first case from Greece. The mycobacterium was isolated from the solid culture (Lowenstein-Jensen slant at 37oC) of a sputum sample after 14 days of incubation. The sample was taken from a 67-year-old male, who was a heavy smoker (1pack/day for 30 years) and had a history of COPD, type II respiratory distress syndrome, diverticulosis and diabetes, and was presented to our hospital with fever (38o C), productive cough, dyspnea, weakness, and acute purpura. The chest radiograph revealed an elevated cardiothoracic ratio, as well as chronic obstructive lung disease, peribronchial infiltrations, consolidations in the right middle and lower lung zones and a small blunt at the left pneumodiaphragmatic angle. The Accuprobe Mycobacterium tuberculosis complex assay (Gen-Probe, San Diego, CA) was negative. Accuprobe also yielded negative results for the Mycobacterium avium complex and Mycobacterium gordonae. Biochemical profile could not distinguish it from other mycobacteria. The banding patterns obtained with GenoType CM and GenoType AS (Hain, Lifescience, Nehren, Germany) were not species-specific [GenoType CM: 1,2,3 and 10, and GenoType AS: 1,2,3 and 12]. The identification of M. thermoresistibile was achieved with the amplification and sequencing of the 16S rRNA gene and the 16S-23S internal transcribed region (GenBank accession: FJ236481). Moreover, a 439-bp fragment of the 65-kDa heat shock protein (hsp65) gene (GenBank accession: FJ236482) was further used for restriction fragment length polymorphism analysis with Hae III (New England Biolabs) and BsteII (New England Biolabs). The BstEII digestion produced two fragments of 235 and 210 bp and the HaeIII digestion produced four fragments of 180, 135, 70 and 50 bp. Although M. thermoresistibile is considered pathogenic, it is rarely isolated from clinical samples. Molecular techniques are essential for its identification.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 131 PP-60

ISOLATION AND FREQUENCY OF Mycobacterium sp IN A GENERAL HOSPITAL DURING A 9-YEAR PERIOD

Portugal C.; Sancho L.; Dias A.; Tancredo L.; Silva M.; Sardinha T.; Sousa Germano Laboratory of Microbiology, Department of Clinical Pathology Hospital Fernando Fonseca – Amadora, Portugal [email protected]

Tuberculosis remains a major public heath problem in Portugal with an incidence rate of 25,3/100.000 inhabitants in 2008, being the majority of the cases in the surroundings of the two major cities (Lisbon and Oporto). Our Hospital is located in the Lisbon’s surroundings and covers a population of 750.000 inhabitants most of them with poor socioeconomic level and immigrants from Africa and East Countries.

Purpose The aim of this study was to investigate the isolation frequency of Mycobacterium sp. in a general Hospital in Amadora, Portugal, during a 9-year period (2000-2008).

Methods A total of 19.417 clinical specimens (15.159 pulmonary and 4.261 extra pulmonary), collected from 9.525 patients, were cultured for mycobateria. All specimens were processed by the NaCl-NaOH method as recommended by CDC, stained by Ziehl-Neelsen, cultured on Lowenstein-Jensen and liquid medium MGIT. Identification was made with the technology Genotype MTBDR plus (HAIN-Lifecience- Germany).

Results Of the 19.417 cultured specimens for mycobateria, 2751 (14,2%) were positive by cultural methods. The positive rates by clinical specimen were: 17% (279/1684) in respiratory specimens and 6% (26/476) in extra pulmonary tuberculosis; 21% (5/23) in pus, 11% (2/14) in biological liquids, 8% (5/66) in biopsy, 6% (7/112) in blood, 5% (1/22) in ascitics fluid, 4% (1/22) in mieloculture, and 3% in urine (4/133) and cerebrospinal fluid (2/84). In 9525 suspected TB patients, 1094 were effective tuberculosis cases (122 TB cases/year average, 97 cases in 2008). The incidence rate by patient was 11,5%. In comparison with the positive results of the culture, Ziehl-Neelsen stain was positive in 39% of the samples (47% of the patients). 96% (1029) of mycobateria isolated were Mycobacterium tuberculosis Complex, 2% (20) M.avium, 2% (20) other species (M.chelonae, M.intracellulare, M.alsiensis / malmoense / szulgai, M.scrofulaceum, M.lentiflavum).

Conclusion In spite of the increase of TB suspected patients (+11,3%), the number of effective tuberculosis cases have decreased (-7,8%) for a 9-year period, which is comparable to 2008 national data (-7,2%) in the last decade. Tuberculosis is a major problem in this area with an recovery rate of 14,2% in the clinical specimens that we receive and in 11,5% of the patients, most of them with Mycobacterium tuberculosis Complex (96%). We have one positive patient every 3 days. In attempt to minimize the impact of this disease, that depend on a large number of factors, we know that the laboratory plays an important role in making a definitive diagnosis in a short time period (ASAP). We also know that is important a close relation between the microbiology laboratory and the hospital doctors and also with the centre that follows the patients in the community (CDP). This is our policy.

132 ESM 2009 PP-61

LABORATORY MICROBIOLOGY CONTRIBUTION TO Mycobacterium spp. DIAGNOSIS IN THREE DISTRICT COUNCILS OF SETúBAL (PORTUGAL), AN AREA WITH HIGH MYCOBACTERIAL INFECTION PREVALENCE.

José Diogo, Ana Rodrigues, Isabel Nascimento, Elisabete Sardinha, Ana Raposo, Rita Figueira, Isabel Monge, Kátia Silva, Maria José Gil, Susana Rodrigues. Laboratory Microbiology, Hospital Garcia de Orta (Almada)

The purpose of this work is to evaluate the incidence of Mycobacterium spp. infection in three district councils of Setubal (Almada, Seixal and Sesimbra) with a high prevalence of this disease, superior to the average in Portugal. The number of patients with mycobacterial infection and positive mycobacteriological study in Laboratório de Microbiologia (LM), Serviço de Patologia Clínica, Hospital Garcia de Orta, E. P. E. (HGO) between 1993 and 2008 were evaluated. Each biological sample was processed as follows: 1) for acid-fast stain with Kinyoun’s carbolfuchsin and microscopic observation with a 100x immersion oil objective; 2) inoculation in Lowenstein-Jensen medium with aerobic incubation at 37ºC and/or inoculation in Middlebrock 7H9 and incubation in MGIT System (Bactec®); and 3) species identification and first line antimycobacterial susceptibility testing. In these sixteen years, LM processed more than 25000 biological products, 3813 were positive and isolated from biological products of 1704 patients. The age, gender, positive samples for BAAR and number of cases of tuberculosis (including variation per year) in Almada, Seixal and Sesimbra was determinated. A relation was established between the disease localization (pleuro-pulmonar, extra-pulmonar and disseminated) by clinical and microbiological criteria. The mean age of the patients was 40,6 years, 1199 (70.3%) were male and 505 (29,7%) were female. Species identification (since 2004) was: 664 (84,1%) M. tuberculosis, 67 (8,5%) M. gordonae, 20 (2,5%) M. avium-intracelular and 39 (4,9%) other mycobacteria. The first line antimycobacterial resistance was determinated in 649 M. tuberculosis strains. The resistance level was: 116 (17,9%) to streptomycin, 76 (11,7%) to isoniazid, 28 (4,3 %) to rifampicin, 17 (2,6%) to ethambutol and 14 (2,2%) to pyrazinamide. The multiresistance (simultaneous resistance to isoniazid and rifampicin) was detected in 27 (4,1 %) strains. Six patients had extensively drug-resistance tuberculosis (XDR-TB). Susceptibility pattern variation per year was also evaluated.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 133 PP-62

Mycobacterium lentiflavum AS A CAUSATIVE AGENT OF ADENOPATHY

Santos, Claudia, Mendes, Ana Constança, Fernandes, Sandra João, Ramos, Maria Helena Centro Hospitalar do Porto - Hospital Santo António

We report the case of a 23 year old female patient, HIV positive, presenting with mesenteric adenopathies of unknown origin. A biopsy was performed and sent for microbiological study. Results turned out negative for aerobic and anaerobic bacterial culture, but with positive AFB examination. Following this information, patient initiated anti-tubercular therapy – Isoniazid, rifabutin, pyrazinamide and ethambutol. Molecular detection of Mycobacterium tuberculosis complex, directly from the clinical sample, was negative. We then performed an in house universal real time PCR targeting a 370 bp region of 16S rDNA bacterial gene, witch gave a positive signal. In order to identify the amplified product, sequencing was performed using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), in ABI PRISM 310 Genetic Analyser (Applied Biosystems). The obtained sequences were analyzed, and identified as Micobacterium lentiflavum. This information led to treatment alteration – (ciprofloxacin, clarothromycin, rifabutin and ethambutol). Conventional mycobacterial cultures (MGIT™ and Lowenstein-Jensen medium) turned out negative after incubation time. Three months later a new biopsy was sent for microbiological study, with positive AFB examination, but negative cultures. Sample was insufficient for molecular study. Sequence based bacterial identification has been widely used to identify microorganisms isolated in clinical samples, particularly when applied to poorly described, rarely isolated or phenotypically aberrant species. We report sequence based bacterial identification of Mycobacterium lentiflavum directly from a clinical sample. Lack of cultural growth did not allow susceptibility testing, and clinical specimen was insufficient for further molecular tests, including testing for mutations associated with antibacterial resistance. However resistance to rifampin has been reported, as has been susceptibility to isoniazid, ethambutol, clarithromycin, amikacin and ciprofloxacin, in vitro susceptibility patterns whose clinical relevance remains uncertain.

134 ESM 2009 PP-63

TEACHING OLD BONES NEW TRICKS; SINGLE NUCLEOTIDE POLYMORPHISM ANALYSIS OF EUROPEAN ARCHAEOLOGICAL M. LEPRAE DNA

Watson, Claire, Lockwood. Diana LSHTM - London School of Hygiene and Tropical Medicine, Keppel Street, London, UK

Background Leprosy was common in Europe eight to twelve centuries ago but molecular confirmation of this has been lacking. We have extracted M. leprae DNA from medieval bones and SNP typed the DNA, this provides insight into the pattern of leprosy transmission in Europe and may assist in the understanding of M. leprae evolution.

Methods and findings Skeletons have been exhumed from 4 European countries (the United Kingdom, France, Denmark and Croatia) and are dated around the medieval period (476 to 1350 A.D.). we tested for the presence of 5 previously identified single nucleotide polymorphisms (SNPs) in 50 aDNA extractions chosen at random from the collection. M. leprae aDNA was extracted from 20 of the 50 bone samples. SNP analysis of these 20 extractions were compared to previously analysed European SNP data using the same PCR assays. Testing for the presence of SNPs in M. leprae DNA extracted from ancient bone samples is a novel approach to analysing European M. leprae DNA and the findings concur with the previously published data that European M. leprae strains fall in to one group (SNP group 3).

Conclusions These findings support the suggestion that the M. leprae genome is extremely stable and show that archaeological M. leprae DNA can be analysed to gain detailed information about the genotypic make-up of European leprosy, which may assist in the understanding of leprosy transmission worldwide.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 135 PP-64

INTERPRETATION OF POSITIVE M. tuberculosis ANTIGEN SPECIFIC IFNΓ RELEASE ASSAYS IN TUBERCULOSIS DIAGNOSIS

Greib Carine 1, Lazaro, Estibaliz 1, Viallard, Jean-François 1, Pellegrin, Jean-Luc 1, Maugein, Jeanne 2 1 - Medecine Interne et Maladies Infectieuses CHU Haut-Leveque Bordeaux 2 - Laboratoire de bactériologie, CHU Haut-Leveque Bordeaux

QuantiFERON-TB Gold in tube test is an accurate test to detect immune responses against active Mycobacterium tuberculosis infection (TB) and has the advantage to eliminate false positive outcomes due to BCG vaccination and non-TB mycobacteria. Howevever there are still some false positive tests which remain unexplained. In this study we aimed to focus on the prevalence and the potential explanation of false positive tests among a cohort of patients accurately screened without TB. A total of 250 adults patients with suspicion of active tuberculosis were enrolled in this study between January 2007 and December 2008. Among them, 88 had positive result in accordance with manufactured interpretation (Nil ≤ 8I/mL, TB antigen ≥ 0.35 IU/ mL and ≥ 25% of Nil value). For 28 patients, tuberculosis was confirmed by culture of Mycobacterium tuberculosis in 26 cases and Mycobacterium bovis in 2 cases. For 9 patients, diagnosis of active tuberculosis was made according to a clinical and paraclinical body of arguments. For 10 patients without active tuberculosis, we could suspect a technical mistake to explain the positive result of the test (TB antigen near 0.35IU/mL for 4 patients and mitogen < 0.5IU/mL for the 6 others). Among the other 41 patients out of 88 with a positive test without any argument for TB, we found that 12 had previous diagnosis of active tuberculosis in the past and 13 came from tuberculosis endemic areas. Finally, 16 positives results out of 88 (18 %) remain without explanation. Errors of diagnosis, bias in collection of medical information (previous or latent tuberculosis infection), or technical mistakes could be possible reasons. In conclusion, quantiFERON-TB is a useful tool for diagnosis of active TB. However the results have to be carefully analysed according to the high rate of false positive diagnosed in this study.

136 ESM 2009 PP-65

DIRECT IDENTIFICATION OF Mycobacterium tuberculosis COMPLEX, Mycobacterium avium COMPLEX AND Mycobacterium kansasii IN SMEAR-POSITIVE CLINICAL SPECIMENS

SUXING WANG, ZHI YU NEO, KE XIN MAK, MARIA DOLORES QUIENG, LI HWEI SNG Central Tuberculosis Laboratory, Department of Pathology, Singapore General Hospital

GenoType@ Mycobacteria Direct (GTMD) was used for direct identification Mycobacterium tuberculosis complex (MTBC), M. avium, M. intracellulare, M. kansasii in 136 specimens from 122 patients. All 136 specimens were AFB smear positive with ranges from 1+ to 4+. The GTMD correctly detected and identified 134 of 136 of the mycobacteria present. Compared to results from culture, this indicates a sensitivity and specificity of GTMD assay of 98.5 and 100%, respectively. These values increased to 100% when specimens with only MTBC isolation were considered. There were two discrepant results during the study. The first was sputum from which M. avium complex was isolated which had been kept at –70oC for nearly 3 years. The test was negative for inhibitors and another sputum collected from same patient at same period was detected as being positive for M. avium by GTMD. This may have been accounted for by degradation of the RNA or a sampling issue. The second was a stool specimen from a HIV-positive patient who had M. avium complex isolated from his stool. Both RNA isolation and amplification were repeated for the same specimen, and the presence of inhibitor was confirmed. Two stool specimens from one HIV-positive patient were initially identified as containing M. avium complex by AccuProbe. GTMD results showed bands matching M. intracellulae and M. kansasii, indicating possible co-infections in this HIV-positive patient. This was confirmed when DNA probe was performed on growth from the LJ slant even though there were no pigmented colonies and both specimens turned out to be positive for M. kansasii. It was most likely that the mixed infection was not detected by routine methods as the M. intracellulare in this case, had outgrown M. kansasii as the predominant organism in broth and solid cultures. Direct identification by GTMD takes about 5 working hours compared to 3 to 5 weeks required for culture isolation and species identification by conventional methods.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 137 PP-66

RAPID DIAGNOSIS AND DRUG SUSCEPTIBILITY TESTING OF TUBERCULOSIS INFECTION: MTD-TEST2 AND BACTEC MGIT 960 SYSTEM

Müllerova, Maria Klinlab Ltd, U vojenske nemocnice 1200, 169 00 Prague 6, Czech Republic, [email protected]

The Czech Republic is a country situated in the heart of the Europe. It has a low incidence of tuberculosis in the last 10 years. However, the increasing number of migrants from countries with high incidence of TB is changing the situation. Samples were collected from patients in the Central Bohemian Region and in a part of Prague (2 millions inhabitants). All samples were tested by MTD-Test2 and conventional methods, partly also by BACTEC MGIT 960, for the detection of M.TB complex. Drug susceptibility tests were performed by conventional methods and BACTEC MGIT 960 (S.I.R.E.+PZA). For the differentiations of separate species of M.TB complex, the GenoType Mycobacteria MTBC test was used. MTD-Test2 for detecting the M.TB complex takes only 3.5 hrs and its performance is highly sensitive and specific. From December 16, 1999 to December 31, 2008 a total of 39,592 different samples were collected for detecting the M.TB complex, comprising of 23,582 sputa, 10,910 bronchoalveolar lavages, 1,492 laryngeal swabs and 3,662 non-pulmonary samples. From 35,930 pulmonary samples were MTD-T2 pos., cult. neg. 416 (1.16%), MTD-T2 neg., cult. pos. 116 (0.32%), MTD-T2 pos., cult. pos. 1,108 (3.08%), and MTD-T2 neg., cult. neg. 34,290 (95.44%). Of the 1,296 isolated strains of M.TB tested for susceptibility to basic AT (S.I.R.E.+PZA), 1,109 (85.57%) strains were susceptible and 187 (14.43%) were resistant for varying combinations of AT; however, further 78 strains (6.02%) were resistant to INH+RIF, i.e. MDR TB. The number of multiresistant patients is rising, especially in the last 3 years: in 2006 10.17% patients were resistant, and from them 4.81% MDR TB, in 2007 8.78% res., 5.36% MDR TB and in 2008 35.51% res., 13.76% MDR TB. These patients are mostly young males, 25-35 years old, from foreign countries. The increase in the number of MDR TB is becoming a problem in our country in view of the need of using alternate AT.

138 ESM 2009 PP-67

FIRST EXPERIENCE WITH GENOTYPE MTBDR ASSAI FOR RAPID EVALUATION OF MDR CASES

Klavdia Levina, Anna Dementieva, Maret Saluotsa North Estonia Medical Centre, Tallin

Tuberculosis (TB) incidence in Estonia decreased from 56.6 per 100.000 population in 1998 till 30.7 in 2008. However, multidrug resistance (MDR) is still very high ~ 12%. Therefore rapid determination of Rifampin (RMP) and Isoniazid (INH) resistance in M.tuberculosis (MTB) isolates remains actual for the initiation of effective chemotherapy to break the transmission of the MDR strains. Drug susceptibility testing (DST) by conventional methods is time-consuming. More rapid results could be achieved by using molecular methods. The goal of our study was to investigate the possibility of application of the Hain Lifescience GenoType MBTDRplus assay as a rapid diagnostic tool for detection of RMP and INH resistance in MTB isolates by comparing the results with those obtained by conventional phenotypic resistance studies. 61 smear positive clinical material and 97 MTB strains recovered from smear negative patients’ specimens by culture have been studied by MBTDRplus assay. Additionally DR was analyzed by standardized DST method on BACTEC MGIT 960 system when culture was later available. Among tested MTB strains, 108 were phenotypically sensitive to INH and RMP, 39 were resistant to both drugs, 9 and 2 have got mono resistance to INH and to RMB correspondently.Wild type patterns were found using the MTBDRplus assay in 107 samples. Specific mutations associated with resistant patterns MTBDRplus assay were displayed in 48 samples. MTBDRplus assay and the DST prepared from strains isolated by cultural methods from smear negative specimens showed 100% agreement between the two methods. Concordant results between MTBDRplus test directly from smear positive samples and conventional DST from later obtained culture strains was found in 95.7 % of cases tested, agreement of the results regarding RMPwas100%. In two (3.4%) smear positive samples resistance-linked mutations were found, but no phenotypically resistance was detected. One strain recovered from AFB positive specimens was INH resistant, but had not displayed mutations conferring INH resistance by MTBDRplus assay directly from the same smear positive specimens. Our results suggest that GenoType MTBDRplus assay is a valuable alternative for rapid detection of resistance and in agreement with the classical methods gives opportunity to break the transmission of the MDR strains.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 139 PP-68

DRUG RESISTANT TUBERCULOSIS IN SLOVENIA AND EVALUATION OF GENOTYPE MTBDRplus TEST IN CLINICAL LABORATORY

Natasa Fajfar, Manca Zolnir - Dovc University Clinic of Pulmonary and Allergic Diseases Golnik, Golnik 36, SI-4204 GOLNIK, SLOVENIA

Slovenia is one of the countries with relative low rate of drug resistant tuberculosis (TB). In the period 1995-2008 the rate was 4.2% (1.6-6.5) and only 0.70 % (0.0-1.8) of Slovenian TB patients had MDR TB in the same period. The aim of our retrospective study was to evaluate the performance of MTBDRplus assay (Hain Lifescinece GmbH, Nehren, Germany) for the detection of rifampicin (RMP) and isoniazid (INH) resistance in comparison to phenotypic drug susceptibility testing method. A total of 48 drug-resistant Mycobacterium (M.) tuberculosis isolates were included in the study. M. tuberculosis drug-resistant strains were obtained from patients living in Slovenia between 1995 and 2008. In total, 20 RMPr/INHr, 27 RMPs/INHr, 1 RMPr/INHs strains were analysed with MTBDRplus assay. The assay was performed according to the manufacturer’s instructions. In comparison to conventional drug susceptibility testing MTBDRplus was able to identify RMP resistance in 21 of the 21 strains (100%), but for INH the accordance of both methods was lower - only in 37 of 47 strains (79%). The most common mutation carried in RMP-resistant isolates was rpoB MUT3-S531L (48%) followed by mutation rpoB MUT1-D516V (33%). In INH-resistant strains dominating mutation was in the gene katG in 60% (MUT1-S315T1 in 68%, MUT2-S315T2 in 29%) and the mutations in the inhA promotor gene in 19%. Comparative analysis demonstrated very good agreement between molecular and phenotypic drug susceptibility results for RMP resistance, but not so for INH. Thus MTBDRplus assay is useful method for the rapid detection of drug resistance, but the results should always be confirmed by the phenotypic methods as well.

140 ESM 2009 PP-69

EVALUATION OF A NEW REAL-TIME PCR KIT FOR THE DIAGNOSIS OF TUBERCULOSIS INRESPIRATORY SPECIMENS

Causse, M; Gutierrez-Aroca, JB; Casal, M Mycobacteria Reference Center. Microbiology Department. Reina Sofia University Hospital, Cordoba (Spain)

Introduction Early diagnosis of tuberculosis is one of the major objectives of the World Health Organization. In its latest update, the Center for Disease Control and Prevention (CDC) recommends the use of a rapid molecular diagnostic technique on at least one sample per patient.

Objectives To evaluate a new kit (COBAS Taqman MTB®, Roche) for the diagnosis of tuberculosis in respiratory samples, and compare results with those obtained by the COBAS Amplicor MTB kit. Culturing (Lowenstein-Jensen or BACTEC MGIT 960) was used for reference purposes.

Material and methods A total of 170 respiratory and 19 non-respiratory samples were processed. Specimens decontaminated were stained with auramine and cultured. A single manual extraction was performed using the AMPLICOR Respiratory Specimen Preparation Kit; eluate aliquots were then amplified using the COBAS Amplicor MTB and the COBAS TaqMan MTB kit. Automatic sample extraction was also performed, using the Ampliprep�� TNAI kit, ��200-µl aliquots being previously incubated at 95ºC for 15 minutes.

Results All 77 smear-positive samples were classified as positive by both kits. Of the 170 respiratory samples tested using the TaqMan, 2 false positives (FP) and one false negative (FN) were recorded. Of the 169 tested using the Amplicor kit, there were 2 FP (different samples than TaqMan) and 2 FN. TaqMan amplification with automatic extraction yielded one false negative and no false positives. Real-time PCR sensitivity and specificity were 98.7% and 97.7% respectively. Use of TaqMan with automatic extraction yielded 98.8% sensitivity and 100% specificity. Kappa indices were 0.95 and 0.97 with respect to the comparator. For smear-negative samples, sensitivity declined to 80% and specificity remained at 97% for the TaqMan kit. For the 19 non-respiratory specimens, all three techniques tested recorded one false negative.

Conclusions TaqMan MTB kit is a real-time PCR method offering the same reliability as the Amplicor MTB kit.�� TaqMan�� MTB kit appeared to display good sensitivity using non-respiratory specimens. It cuts down time-to-results from 6 to 2.5 hours Automatic extraction reduced the number of false positives and considerably shortened handling time.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 141 PP-70

QUANTIFERON-TB GOLD ASSAY (QFT) AND TUBERCULINE SKIN TEST (TST) CLINICAL PERFORMANCE FOR THE DIAGNOSIS OF ACTIVE TUBERCULOSIS

Karabela Simona, Papaventsis Dimitrios, Nikolaou Stavroula, Konstantinidou Efthimia, Sainti Asimina, Ioannidis Panayotis, Kanavaki Sofia National Reference Center for Mycobacteria, “Sotiria” Hospital, Athens, Greece

Objective The purpose of this study was to evaluate and compare Quantiferon-TB Gold In-Tube (QFT, Cellestis, Australia) and the tuberculine skin test (TST) in patients with active TB, with and without previous BCG vaccination.

Methods Patients with symptoms compatible with active TB were included. The TST was performed according to the Mantoux method and the QFT assay according to the manufacturer’s instructions. The cut-off value for a positive result was ≥0.35 IU/ml interferon-gamma (IFN-γ). Sensitivity, specificity, positive and negative predictive values were calculated and compared for QFT and TST tests. Agreement between QFT and TST was assessed by the kappa (κ) coefficient.

Results A total of 296 patients were enrolled in the study. One hundred eighty-nine had a record regarding BCG vaccination. Forty-four (23%) of the 189 patients had been vaccinated. In total, the sensitivity and specificity of QFT, excluding those with indeterminate results, was 79% (52/66; 95% CI: 66-88%) and 66% (153/167; 95% CI: 58��-��73��%), respectively. The sensitivity and specificity of TST was 72% (46/64; 95% CI: 58-83%) and 67% (156/232; 95% CI: 59-74%), respectively. The overall concordance between the QFT and TST tests was 70.2%, with a kappa value of 0.46 (95% CI: 0.289-0.524). In the BCG- vaccinated subgroup, agreement between the two assays was 66%, with a kappa value of 0.350 (95% CI: 0.103-0.597). The difference with the non-vaccinated subgroup (κ=0.463; 95% CI: 0.303-0.623) was considered to be not quite statistically significant (p>0.05). Initial TST positive screening followed by a QFT positive result was found to have greater sensitivity and specificity in the non-vaccinated [sensitivity=79% (95% CI: 59-92%); specificity=81% (95% CI: 71-88%)] compared to the BCC-vaccinated subgroup [sensitivity=67% (95% CI: 30-92%); specificity=75% (95% CI: 57-89%)].

Conclusion This study confirmed previous reports that QFT assay has higher sensitivity for detecting active TB compared to TST. An overall moderate agreement between TST and QFT was found. The difference in agreement between non-vaccinated and BCG-vaccinated subgroups could be attributed to TST influence by vaccination. In patients with active TB and no BCG-vaccination history, TST screening followed by subsequent QFT testing proved to present the highest sensitivity and specificity for TB diagnosis. Larger prospective studies are needed to confirm our results.

142 ESM 2009 PP-71

CLINICAL PERFORMANCE OF QUANTIFERON-TB GOLD ASSAY (QFT) FOR THE DIAGNOSIS OF LATENT TUBERCULOSIS IN DIFFERENT PATIENT GROUPS

Objective The purpose of this study was to evaluate the performance and usefulness of Quantiferon-TB Gold In-Tube (QFT, Cellestis, Australia) in the diagnosis of latent tuberculosis and to compare it with the tuberculin skin test (TST).

Methods A cohort of 395 high risk adults was prospectively evaluated. Study groups consisted of 139 neoplastic patients, 98 with autoimmune diseases (SLE, rheumatoid and psoriatic arthritis), 20 immunossupressed (e.g. HIV, hemodialysis) and 26 Intensive Care Unit (ICU) patients, and 112 patients with no underlying disease. The TST was performed according to the Mantoux method and the QFT assay according to the manufacturer’s instructions. The cut-off value for a positive result was ≥0.35 IU/ml interferon-gamma (IFN-γ). QFT and TST were simultaneously performed in 297 patients.

Results Overall, QFT gave a positive result in 72/395 (18.22%) patients. Among the different risk groups, ICU and immunosuppressed patients represented the highest positive rates (19% and 20%, respectively). Indeterminate results (7% on the whole) were more often seen in ICU (34.6%), neoplastic (7.2%) and in patients with autoimmune disease (7.2%). Indeterminate results represented <1% in patients with no underlying disease. Strength of agreement between GFT and TST results was poor (agreement=55.44%, kappa=0.171; 95% CI: [0.064-0.278]).

Conclusion QFT is a very usefully method in TB diagnosis because in contrast to TST, it reduces over diagnosis of latent TB in previously BCG vaccinated, distinguishing truly tuberculosis cases from BCG vaccinated individuals and/or non-tuberculous infections. As a consequence, QFT provides valuable information for therapeutical decision-making.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 143 PP-72

TUBERCULOSIS DIAGNOSIS BY QUANTIFERON TB GOLD ASSAY IN AREAS WITH DIFFERENCES IN TB INCIDENCE

Nikolaou Stavroula, Karabela Simona, Papaventsis Dimitrios, Sainti Asimina, Konstantinidou Efthimia, Ioannidis Panayotis, Kanavaki Sofia National Reference Center for Mycobacteria, “Sotiria” Hospital, Athens, Greece

Objective Evaluation of Quantiferon TB Gold in Tube method (QFT) for the latent TB diagnosis in patients originated from countries with high (group A) and low (group B) incidence of TB.

Material 948 whole blood samples from 153 (16,15) individuals belonging to group A and 795 (83,9%) to group B.

Method Performance of Quantiferon TB Gold in Tube (Cellestis, Australia) method according to the manufacturers’ instructions.

Results QFT positive results was detected in 93/153(60.8%) individuals of group A and 273/795 (29,35) of group B (p<0,0001). Clinical information for previous BCG vaccination was available in 351 cases: 65 of group A and 286 of group B, where QFT confirmed latent TB in 36/65(50,8%) and 46/286(16,1%) of group B (p<0,0001). As it was expected, Tuberculin Skin Test (TST) was positive in all 351 cases with previous BCG vaccination.

Conclusion QFT is a highly diagnostic and useful method, especially in patients originated from areas with high incidence of TB. In contrast to widely used TST, it reduces overdiagnosis of latent TB in previously BCG vaccinated.

144 ESM 2009 PP-73

QUANTIFERON® -TB GOLD IN-TUBE TEST USED IN PRAGUE PATIENTS LISTED IN THE NATIONAL TUBERCULOSIS REGISTER

Havelkova, Marta 1, Bartu, Vaclava 2, Kubin, Milan 3 1 - National Institute of Public Health – NRL for mycobacteria 2 - Charles University , Faculty of Medicine, Faculty Thomayer Hospital 3 - Prague Hygiene Institute

In 2006-2007, 65 (29.0%) of 224 patients registered in the A15, A16 and A18/19 categories (58.5%, 32.3% and 9.2% of all patients, respectively) were assessed using both the QuantiFERON – TB Gold In-Tube (QFT) method and the tuberculin skin test (TST) with 2 TU PPD. After stimulation with tuberculosis-specific antigens, a low level of interferon-gamma (IFG) production (< 0.35 IU) was found in 18 (27.7%) patients and moderate or high levels (> 0.35 IU) in the remaining 47 (72.3%) patients. After incubation with a non-specific mitogen, a low level of IFG production was recovered in 13 (20%) individuals, with moderate or high levels being found in the remaining 52 (80%) patients. The TST revealed low levels of skin infiltrate reaction (0-5 mm) in 25 patients (39.1%) and moderate or high levels of skin reaction (range 6 - > 30 mm) in the remaining 39 individuals (60.9%). The high proportion of low reagent levels in both the QFT and tuberculin skin tests can be explained by a high number of older patients, comorbidity with malignant tumours, diabetes or general fatigue in pre-terminal patients, and the immune

systém insufficiency related to these factors. ^ This presentation was fully supported by project KAN 200520702 of the Grant Agency of Czech Academy of Sciences (GAAV CqR).

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 145 PP-74

PRACTICAL EXPERIENCE OF USING A DNA AMPLIFICATION ASSAY FOR RAPID DETECTION OF Mycobacterium tuberculosis COMPLEX IN RESPIRATORY SPECIMENS

J. Cacho1, A. García-Cañas1, A. González Torralba1, I. Cano2, A. Pérez Meixeira3, A. Ramos Martos2, and M. Sánchez-Concheiro1 1 - Servicio de Microbiología, Hospital Universitario de Getafe, Madrid 2 - Servicio de Neumología, Hospital Universitario de Getafe, Madrid 3 - Servicio de Salud Pública, Comunidad de Madrid, Madrid.

Objectives To evaluate experience in a clinical microbiology laboratory of using a DNA amplification assay for routine detection of Mycobacterium tuberculosis complex (MTC) performed once weekly, and to compare this method with microscopy and culture.

Methods A total of 507 respiratory specimens from 419 patients were screened for tuberculosis (TB). Smear examinations, culture and polymerase chain reaction (PCR) were performed on each sample. Specimens were processed according to standard laboratory protocols. All samples were processed exactly as described in Cobas Amplicor MTB Methods Manual (Roche Molecular Systems, USA). This method was performed once per week. The following two groups of samples were considered to be true positive: (1) samples which were culture positive for MTC; and (2) all samples which were culture negative for MTC but positive to PCR, provided that one or more of the following criteria were met: (i) the samples originated from a patient whose other samples were culture positive; (ii) the patient’s clinical history provided evidence of TB sufficient to warrant initiating treatment for TB.

Results Inhibition of PCR was seen in 15 (2.9%) specimens. A total of 37 (7.3%) samples were considered to be true positive: 33 samples grew MTC in culture and 4 were culture negative for MTC but positive for PCR and met the criteria as described previously. The overall sensitivity and specificity of PCR as compared to true positive samples was 83.8% (31/37) and 99.1% (466/470), respectively; that of culture 89.2% (33/37) and 100% (470/470), respectively; and that of direct microscopy 64.9% (24/37) and 99.8% (469/470), respectively. The average time to reporting for true positive samples was 6 days for positive PCR and 11.4 days for positive culture. Of the smear-positive, true positive samples, 91.7% (22/24) were PCR positive. Of the smear-negative, true positive samples, 69.2% (9/13) were PCR positive. The 9 samples classified as true positive and smear negative were from 9 different patients. Treatment was started earlier in 44.5% of these patients because positive PCR findings were obtained. The average time to reporting was 5.4 days for positive PCR findings and 17.4 days for positive culture findings.

Conclusion MTC infection was confirmed for PCR in 91.7% of smear-positive specimens. Although PCR was performed once weekly, treatment in 44.5% of patients was initiated earlier because of positive PCR results from smear-negative samples.

146 ESM 2009 PP-75

REAL-TIME POLYMERASE CHAIN REACTION FOR THE DIRECT DETECTION OF MYCOBACTERIUM TUBERCULOSIS IN CLINICAL SPECIMENS

Karen, Morgan Joint Clinical Research Centre, Kampala, Uganda

Introduction The resurgence of tuberculosis is a leading cause of death worldwide. Since M. tuberculosis, is slow growing, methods of diagnostic testing based on culture are delayed. This study describes the development of a real-time polymerase chain reaction (RT-PCR) assay and subsequent clinical testing.

Methods Patients were recruited from routine TB suspects in the Monterey County Public Health laboratory, CA, USA. Initially, using Bactec MGIT 960 cultures as the gold standard, 231 respiratory specimens composed of 76 specimens from culture-confirmed tuberculosis cases and 155 culture negative specimens were analyzed by RT-PCR. Over the next 2 years 206 respiratory patient specimens were tested from 81 flurochrome smear AFB positive and 125 negative sputa. DNA extraction was performed directly from patient specimens by the modified Qiagen mini-Amp kit (Valencia, CA). The RT- PCR was performed on the Roche LightCycler instrument (Mannheim, Germany). The assay was designed to target the ITS region of the 16S rRNA gene of M. tuberculosis using Taqman hybridization probes for detection of amplicons.

Results The developed RT-PCR assay yielded results in one day and achieved a sensitivity of 85.5% and specificity of 100%. In subsequent clinical use over the two year period the RT-PCR assay achieved a sensitivity of 92.3% and specificity of 100% in direct detection of MTB from 206 respiratory patient specimens,while in contrast fluorochrome smears achieved only a sensitivity of 60.5%% for non-specific AFB detection in the same population. The RT-PCR assay detected MTB in 64.7% of the smear negative but culture confirmed patient specimens. The RT-PCR assay costs $14 per test, inexpensive compared to commercial MTB assays ($60).

Conclusion RT-PCR methodolgy can be used to detect MTB directly in patient specimens within eight hours, without waiting for culture growth. This rapidly increases and enhances specific detection and treatment of MTB.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 147 PP-76

THE UTILITY OF MOLECULAR TESTING IN ROUTINE MYCOBACTERIOLOGY DIAGNOSIS

Fanourios Kontos, Loukia Zerva. Clinical Microbiology Laboratory, Medical School of Athens, “Attikon” University Hospital, Athens, Greece.

Objective Molecular methods increasingly replace phenotypic tests in Mycobacteriology. This study describes a “molecular” strategy that was developed for routine testing of clinical samples and isolates with the goal of shortening turnaround time (TAT) and providing accurate results.

Methods All samples submitted for mycobacterial cultures (from 12/2006 to 3/2009) were processed and cultured by standard methodology. The first smear (+) specimen of any patient and every first (+) culture were tested by an in-house IS6110- PCR in order to differentiate between Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM) (TAT 4 hours). IS6110-PCR (+) specimens or cultures were tested by Genotype MTBDRplus (Hain-LIFESCIENCE) for MTBC species confirmation and detection of Isoniazid and Rifampicin resistance (TAT 2 days). Subsequent culture (+) specimens from the same patient were tested by Accuprobe MTBC (Biomerieux) (TAT 2 hours). The susceptibility testing of MTBC isolates was performed by MGIT960 (Becton Dickinson). If a (-) result was obtained by the IS6110- PCR, identification of isolates proceeded using both Genotype Μycobacterium CM and AS (Hain-LIFESCIENCE) (TAT two days). For NTM species confirmation a PCR-RFLP analysis of the hsp65 gene was applied (TAT 3 days); additionally, sequencing of the 16S rRNA gene (TAT 5 days) represented the reference identification method for selected isolates.

Results Out of 4.000 specimens, 160 were culture positive (4%) including 85 MTBC positives (63.5% acid fast stain [AFS] positive) and 75 NTM positives (26.7% AFS positive). Five samples contained more than one NTM species, which were identified only by molecular testing. There was complete agreement between all molecular identification methods; however 16S rRNA sequencing was more informative (examples: M. fortuitum and M. lentiflavum, M. celatum). All MTBC isolates were Rifampicin susceptible, two were high- and one low-level Isoniazid resistant by MGIT960; only the latter was not recognized by Genotype MTBDRplus.

Conclusions Focusing on molecular methodology resulted in fast and accurate final reporting. The low incidence of MTBC positivity among tested specimens (2,1%) justified the decision not to use indiscriminately a direct molecular test for tuberculosis diagnosis. The frequent occurrence of NTM (48,5% of all culture positive samples) necessitates direct molecular testing of all AFS (+) specimens.

148 ESM 2009 PP-77

DETECTION OF Mycobacterium tuberculosis DNA IN FORMALIN-FIXED, PARAFFIN-EMBEDDED TISSUE SPECIMENS BY SPOLIGOTYPING: APPLICATION TO HISTOPATHOLOGICAL DIAGNOSIS.

Salas S1, Hernández J1, Ojeda P2, Awad C2, de la Hoz F3, Murcia MI1. 1 - Departamento de Microbiología, Facultad de Medicina. Universidad Nacional de Colombia. Bogotá, Colombia 2 - Hospital Santa Clara E.S.E., Bogotá, Colombia 3 - Departamento de Salud Pública, Facultad de Medicina. Universidad Nacional de Colombia. Bogotá, Colombia

In Colombia, Tuberculosis (TB) remains as a public health problem with an incidence of 25 per 100 000 population while extra-pulmonary TB has increased. The Spoligotyping method has demonstrated to be a good tool to improve the TB extra-pulmonary diagnosis.

Purpose of study To identify DNA of Mycobacterium tuberculosis from Formalin-fixed paraffin embedded Tissues specimens (FFPET).

Methods We examined 160 FFPET storaged for 13 years and with a suspected diagnosis of Tuberculosis infection according to histopatological analysis. Spoligotyping was carried out as previously described with some modifications. DNA extraction with CHELEX was used and human DNA was negative control. The Spoligotype films were scanned and classified by using GeneTools software followed by manual editing and confirmation. Statistical analysis was performed using SPSS V.15.0.

Results Of the 160 samples analyzed by Spoligotyping, 78 (48.8%) cases showed absence of signal at the spot 33 to 36 compatible with M. tuberculosis. Nevertheless SPOL-DB4 was made with DNA obtained from cultures, we in tented to compare our patterns obtained and we founded only 4 samples compatible with pattern previously described LAM (2), U (1) and Haarlem lineage (1). Incomplete patterns were observed in 34 (21.2%) and no patterns were obtained in 48 (30.0%). Negative controls did not yield any spoligopatterns. Exact Fisher’s statistic showed a significant difference between the positivity of Spoligotyping and storage time (P <0.05) while for the kind of tissue found no differences (P> 0.05).

Conclusions In 48.8% cases we confirmed the diagnosis of M. tuberculosis. The spoligotyping method is a tool that provides some information about FFPET, the results of this can be affected by storage time. In order to obtain better results is necessary to examine the samples immediately after her obtention.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 149 PP-78

Pott´s Disease: an ancient disease?

Cardoso, Sara, Coelho, Rui, Paulo, Cristiana, Abreu, Candida, Silva, Susana, Gomes, Helena, Sarmento, António Hospital S.João

Introduction Skeletal tuberculosis is a rare disease in developed countries although it´s still a significant cause of disease in Portugal due to the high prevalence of tuberculosis and an insidious and non-specific presentation of this form of disease. Clinical report: We report 3 cases of Pott´s disease.

Case 1: Male, 62y. History of multiple sclerosis under imunomodulator and leg trauma with disability. He went several times to the emergency room due to intense lombalgy without trauma and discharged with non-steroidal anti-inflam- matory (NSAIs) medication, without relief. D12-L1 fracture was diagnosed and the patient was discharged under con- servative treatment. Fifteen days later, he appeared with paraparesia, fever, asthenia and weight loss and was submitted to surgery. Histology of the bone fragments revealed acid fast bacilli. The direct and cultural exam of gastric lavage (GL) were positive for M.tuberculosis complex (MTC). Thorax X-ray (XR) was normal.

Case 2: Female, 88y. History of previous ribs and femur trauma fractures. She had complaints of lombalgy and progressive paraparesia during the last year and was chronically medicated with NSAIs. Three months before admission anorexy and weight loss appeared. She had no fever. The magnetic ressonance revealed D6-D7 vertebral body fracture with cavitation and medular compression. She was submitted to surgery. The bone cultural exam was positive to MTC. The thorax XR was suggestive of pulmonary involvement but the GL mycobacteriology exam was negative.

Case 3: A 77y woman with past history of pulmonary tuberculosis and recent history of dorsal trauma with D12 fracture, treated conservatively. Later she was admitted with paraplegia and submitted to surgery. The bone culture, bronchoalveolar lavage culture and DNA (polymerase chain reaction) were positive for MTC. The cerebrospinal fluid had 24cells/ uL, high ADA (123U/L), proteins (10,20g/l) and low glucose (0,14g/l), but DNA for MTC and culture were negative. All three patients were treated surgically and medically with rifampin, isoniazid, ethambutol and pyrazinamide. All of them had come with dorsal or lombar pain and progressed to paraparesia and plegia, which showed some improvement with antibiotics and physiotherapy.

Conclusion Skeletal tuberculosis is a silent re-emergent disease, for which our doctors are not aware, leading to delayed diagnosis and causing severe neurological damage like Pott´s paraplegia as it was observed in our patients.

150 ESM 2009 PP-79

OSSEOUS TUBERCULOSIS AT AGE OF 9 MONTHS

Loureiro, Carla 1, Matos, Gabriel 1, Balacó, Inês 1, Mota, Marta 2, Nogueira, Célia 2, Lemos, Sónia 1, Rocha, Graça 1 1. Pediatric Department/H. Pediatrico, CHC 2. Mycrobiology Laboratory, Coimbra Medicine Faculty

Background and Aims Osseous tuberculosis is rare, mainly as a primary disease in a previously healthy infant. Several mycobacteria may be involved such as M. tuberculosis, M. bovis and M. avium. When there is no association with a primary pulmonary disease in a child vaccinated with Calmet-Guérin Bacillus, M. bovis may be the causal agent.

Case Report A previously healthy 11 month-old infant BCG vaccinated at birth was admitted with a 2 month evolution of a right elbow tumefaction. The X-ray presented destruction of the trochea, severe periostic reaction and soft tissue tumefaction, and the ecography a fluid-filled cavity. MRI suggested neuroblastoma or Ewing sarcoma. Sedimentation rate and specific eno- lase were elevated. On surgery a whitish soft mass associated with necrotic fluid compressing the cubital nerve was found. The histological examination revealed characteristic features of caseum and allowed the diagnosis of osseous tuberculosis. Mycobacterium tuberculosis complex was identified by Real-Time PCR in osseous biopsy. Gastric fluid and urine were negative. Our patient recovered after surgical debridement and combination drug therapy. He developed a local cutaneous fistula (no agent identified on fluid culture) and posterior soft tissue calcification. At 22 months age he remains with no other relevant infections.

Conclusions Skeletal tuberculosis with extravertebral location is rare. Tuberculous osteomyelites of the limb bones requires a high index of clinical suspicion along with radiological and histopathological investigation in order to establish the diagnosis. Tuberculosis is still an important differential diagnosis in unusual bone conditions

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 151 PP-80

DIFFERENCES IN DIRECT ANTITUMORAL CAPACITY AMONG THE VARIOUS Mycobacterium bovis BCG SUBSTRAINS

SP Secanella, M Luquin, E Julián Dept. Genètica i Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra (Barcelona)

The administration of Mycobacterium bovis Bacillus Calmette-Guérin (BCG) is the first treatment option for avoiding the recurrence of bladder cancer. Various BCG substrains are used. These substrains differ genetically and display differential antigenic determinants, which it has been suggested may influence the efficacy of BCG as vaccine in tuberculosis studies. In tuberculosis, evolutionary early strains are more efficacious than are the more attenuated evolutionary late strains. The impact of these differences on BCG antitumoral capacity in bladder-cancer cell lines has not been addressed. We aimed to compare the direct antitumoral activity of different BCG substrains by inhibiting cell proliferation and by triggering the production of cytokines in bladder-cancer cell lines. T24, J82 and RT4 ��h��uman bladder-cancer cell lines were cultured with different doses of BCGs. We tested three evolutionary early BCG substrains, Japan, Moreau and Russia, and five evolutionary late strains, Connaught, Danish, Glaxo, Phipps, and Tice. Inhibition of cell proliferation was assessed by using a colorimetric assay at different time points; the production of interleukin (IL)-6 and IL-8 was measured in cell culture supernatants using enzyme-linked immunosorbent assay. Among the different BCG substrains, in the case of T24 and J82 cell lines, Connaught and Russia induced both the highest inhibition of proliferation and cytokine production. In contrast, Glaxo and Phipps (for the T24 cell line) and Glaxo and Tice (for the J82) were the least efficacious both in reducing cell viability and in inducing cytokine production. The remaining BCGs behaved differently depending on the cell line. Finally, for the RT4 cell line, all BCG strains inhibit cell proliferation at the same level, except for Danish and Glaxo, which were seen to be less efficacious. As regards cytokine production, IL-6 production was not detected in any culture, whereas low levels of IL-8 production were observed, the lowest being for Danish and Glaxo cultures. The results showed that Connaught and Russia are the most efficient BCGs and that Glaxo is the least effective, both in the inhibition of tumoral-cell proliferation and the induction of cytokine production. No correlation was observed between BCG antitumoral efficacy and genotypic evolutionary classification.

152 ESM 2009 PP-81

ROLE OF TNF-a GENE POLYMORPHISMS IN HOST GENETIC SUSCEPTIBILITY TO PULMONARY TUBERCULOSIS

Anoosheh, Saber 1, Farnia, Parissa 1, Noruzi, Jamileh 2, Kargar, Mohammad 3, Kazempour, Mehdi 1, Seif, Shima 1, Masjedi, Mohammad Reza 4, Velayati, Ali Akbar 4 1 - Mycobacteriology Research Center, NRITLD, Shahid Beheshti University (M.C) 2 - Microbiology Department, Iran University of Medical Science 3 - Microbiology Department, Jahrom Azad University 4 - National Research Institute of Tuberculosis and Lung Disease, Shahid Beheshti University (M.C)

Preface and goal Tuberculosis is one of most common infectious diseases and it causes death of more than 3 million people a year, worldwide. It caused by Mycobacterium tuberculosis and approximately one - third of world population are infected with this bacteria, but only 5 - 10 % of them develop active TB. Therefore, individual differences in susceptibility to TB are expected. These differences might be due to host factors especially genetic diversity between populations. TNF-α as a pro-inflam- matory cytokine, play a key role in host defense against tuberculosis. Presence of mutation in this gene can influence the effectiveness, performance and capability of immune Responses against infection. The Aim of this study was to investigate the frequency of TNF-α alleles and relationship between susceptibility to TB and TNF-α gene variations.

Materials and methods

A case-control study was conducted and 65 healthy controls and 65 TB patients were enrolled. Genotype of TNF-238,

TNF -244, TNF-308, TNF -857 and TNF-863 were distinguished using by PCR-RFLP method. The results were analyzed by SPSS v.16, Fisher exact and Hardy-Weinberg tests.

Results

Obtained results showed that, TNF-308, TNF -857 and TNF-863 were as a high frequency mutation regions in population levels, and also we found a significant differences at TNF-308 and TNF -857 between two group of controls and patients ( P-value < 0.05 ).

Conclusion presence of mutation in TNF-308 and TNF -857 regions probably increases host susceptibility to mycobacterial infection and Genotyping of these regions can be used for screening of high risk persons. Also according to high frequency distribution of mutations in TNF -857 and TNF-863 regions, further studies on association of these regions is suggested.

Key words Tuberculosis, Genetic Susceptibility, Cytokines, Gene Polymorphism

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 153 PP-82

MYCOLACTONE INTERFERES WITH THE PROTECTIVE IFN-g-DEPENDENT ACTIVATION OF MACROPHAGES DURING INFECTION WITH Mycobacterium ulcerans

Egídio Torrado1; Alexandra G. Fraga1; Elsa Logarinho1; Teresa G. Martins1; Jenny A. Carmona1; José B. Gama1; Maria A. Carvalho2; Fernanda Proença2; António G. Castro1; Jorge Pedrosa1 1 - Life and Health Sciences Research Institute (ICVS). School of Health Sciences, University of Minho. Braga, Portugal 2 - Chemistry Research Center. School of Sciences. University of Minho. Braga, Portugal

Mycobacterium ulcerans is the etiological agent of a necrotizing cutaneous disease, known as Buruli ulcer. The pathology caused by this pathogen is associated with the production of the lipidic exotoxin mycolactone. Following our recent demonstration of an intramacrophage growth phase for M. ulcerans, we investigated the biological relevance of interferon-gamma (IFN-γ), as well as the mechanisms activated by this cytokine in M. ulcerans-infected macrophages. We used three different M. ulcerans strains selected based on their virulence for mice and the type of mycolactone produced: the low virulent mycolactone-negative strain 5114; the intermediate virulent, mycolactone C-producing strain 94-1327; and the highly virulent, mycolactone D-producing strain 98-912. IFN-γ-deficient mice showed an increased susceptibility to infection only with strains 5114 and 94-1327, suggesting that this cytokine plays a protective role in infections with the low and intermediate virulent strains of M. ulcerans, but not with the highly virulent strain. In line with this, IFN-γ-activated mouse primary bone marrow-derived macrophages controlled the proliferation of the low virulent and the intermediate virulent strains, the latter only at low multiplicities of infection. The effector mechanisms induced by IFN-γ in infected macrophages leading to M. ulcerans growth restriction involved both phagosome maturation and acidification, as well as increased nitric oxide production. In agreement, the addition of mycolactone D, purified from cultures of the highly virulent strain, led to a dose-dependent inhibition of phagosome maturation and nitric oxide production in IFN-γ-activated cultured-macrophages infected with the mycolactone- negative strain, resulting in an increased bacterial burden. Our results suggest that the protection mediated by IFN-γ during the intramacrophage phase of M. ulcerans infection depends on the type and amount of mycolactone produced.

154 ESM 2009 PP-83

VIRULENCE, IMMUNOGENICITY AND PROTECTION INDUCED BY ´Mycobacterium habana´ STRAINS IN A MURINE MODEL OF PULMONARY TUBERCULOSIS.

Montoro, Ernesto 1; Valdés, Iliana 1; Aguilar, Diana 2; Orozco, Hector 2; Hernández-Pando, Rogelio 2 1. Institute of Tropical Medicine “Pedro Kourí”(IPK), Havana, Cuba 2. National Institute of Medical Science and Nutrition ¨Salvador Zubirán¨. Mexico DF, Mexico

Mycobacterium habana’ was first isolated in Cuba by Valdivia, in 1971. Later, was demonstrated its protection capacity against M. tuberculosis and other mycobacteria. We studied the virulence, immunogenicity and protection of 3 strains of ‘M. habana’ using Balb/c mice. The first experiment was done to know the virulence potential of ‘M. habana’ using a progressive pulmonary TB model. In the 2nd assay mice were vaccinated with 3 doses of bacilli. The grade of immunogenicity was related with the induction of IFNγ by the stimulation of the main organs with antigens of M. tuberculosis. The best doses that induced immunogenicity were used in the 3rd experiment for animal vaccination. Two months later mice were challenged with M. tuberculosis H37Rv and Beijing genotype. All the animals infected with M. habana TMC-5135 and IPK-337 were alive until the end of the experiment. IPK-220 strain showed about 20% of death to the seven week post- infection. All the strains had significative differences when we compared with the control group infected with H37Rv strain. The values of the colony forming units (CFU) were in correspondence with the survival rate. The percentage of pneumonia was higher for ‘M. habana’ IPK-220, showing final values similar to mice infected with H37Rv strain. Due to the virulent behaviour of ’M. habana’ IPK-220 we discharged it for the coming assays. The most important results of the 2nd experiment were the statistical differences in the IFNγ production founded in groups vaccinated of ‘M. habana’ IPK-337 and TMC-5135 strains, respectively, with the BCG group. The lung CFU for these doses showed a decreasing tendency with a total sterilization to the final of the experiment. Animal vaccinated with ‘M. habana’ strains and challenged with M. tuberculosis H37Rv had the highest survival. These results are in accordance with the low percentage of pneumonia with both stains. Nevertheless this differences was not statistical significative in comparison with BCG group. With the Beijing challenge we observed differences between vaccination with TMC-5135 and BCG group. The lungs of the animals that received BCG and challenged with Beijing showed more than 70% of pneumonia and a lower granuloma area. The CFU reveals lower lung bacilli load in mice vaccinated with ‘M. habana’ strains. The final results demonstrate the potential of ‘M. habana’ to protect against TB infection.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 155 PP-84

DENDRITIC CELLS DIFFERENTIALLY EXPRESS IL12-FAMILY CYTOKINES AFTER INFECTION WITH Mycobacterium tuberculosis OR M. bovis BCG

Margarida Saraiva, Carole Sousa, Jenny A. Carmona, Andrea Cruz, Jorge Pedrosa, A. Gil Castro Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal

IL-12 family is a group of heterodimeric cytokines, composed of 4 related cytokine members: IL-12, IL-23, IL-27 and IL-35. These cytokines, that share some functions and receptor components of IL-12, act on CD4+ T cells modulating the type of T helper and T regulatory responses, and initiating the development of the acquired T cell response to intracellular pathogens, such as Mycobacterium tuberculosis (MTb) or M. bovis BCG. Bone marrow derived dendritic cells (BMDC) originating from wild-type mice were exposed to live MTb strain H37Rv or to BCG for different periods of time. The kinetics of mRNA production of each monomer that form the cytokines of the IL-12 family (p19, p28, p35, p40, Ebstein- Barr-Virus-induced gene 3 (Ebi-3)) was determined. We show that MTb-stimulated BMDC were strong producers of p40, p35 and p19, whereas BMDC exposed to BCG expressed much lower levels of these cytokines. The main TLR involved in the recognition of MTb and BCG and activation of BMDC is TLR2, since in its absence the expression of the various monomers was nearly abrogated. A consequence of this differential activation of BMDC was reflected on the distinct type of T helper responses developed when MTb- or BCG-infected BMDC presented OVA peptide to TCR-transgenic CD4 T cells. MTb-infected BMDC were able to induce the development of both Th1 and Th17 responses, whereas BCG- infected BMDC induced Th17 responses. We are currently addressing the molecular mechanisms that differentiate the response of BMDC in the context of an MTb or a BCG infection. Understanding the details of DC activation by MTb or BCG and its consequences on the CD4+ T cell arm of the immune response will help to reveal important aspects to be improved for a better vaccination strategy.

156 ESM 2009 PP-85

ANALYSIS OF M. smegmatis MUTANTS RESISTANT TO MS6 INFECTION

Simões, Marta Filipa; Jordão, Luísa; Teles, JMM; Couto, S; Moniz-Pereira, José; Pimentel, Madalena Centro de Patogénese Molecular-Unidade dos Retrovirus e Infecções Associadas, Faculty of Pharmacy, University of Lisbon, Portugal

Mycobacteriophages represent excellent model systems for studying mycobacterial hosts. Ms6 is a temperate myco- bacteriophage that infects Mycobacterium smegmatis. The first step of a dsDNA phage infection is adsorption to a host receptor followed by injection of DNA into the cytoplasm. Characterization of the phage resistant mutants represents a common genetic strategy for the identification of mycobacterial genes involved in the synthesis of parietal phage receptors. The study of these genes is of highly importance, as phage receptors are also involved in the entry of others molecules, such as antibiotics, into the cell. In order to identify the Ms6 receptor we started to select from a mutant library obtained after a transposition event using the pCG79 system (Guilhot et al, 1994), M. smegmatis mutants to a Ms6 infection. DNA obtained from these mutants was submitted to an enzymatic restriction analysis, which allowed the selection of four mutants with different profiles. In order to understand which step of the phage infection was affected, adsorption and infection assays with DAPI stained phages were performed. We found different profiles among the selected mutants suggesting that different steps of the infection are affected. With these results, our goal is to identify the mycobacterial genes disrupted by the transposition event. Identification of the affected genes is an important achievement which may contribute to the characterization of the phage receptor. Guilhot, C., I. Otal, I. Van Rompaey, C. Martín, and B. Gicquel. 1994. Efficient transposition in mycobacteria: construction of Mycobacterium smegmatis insertional mutant libraries. J. Bacteriol. 176: 535-539.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 157 PP-86

HUMORAL RESPONSE IN TUBERCULOUS PATIENTS AGAINST THE MYCOLIC ACIDS OF Mycobacterium tuberculosis

Julián, Esther Gómez 1; Rodríguez-Güell, E 1; del Val-Romero, B 2; Clivillé, R 2; Cañete, C 3; Navarro, A 3; de Gispert, FX 3; Luquin, M 1; Alonso, C 2 1 - Dept. Genètica i Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra (Barcelona) 2 - Dept. Microbiologia, Consorci Sanitari Integral, L’Hospitalet de Llobregat (Barcelona) 3 - Unitat de Respiratori, Consorci Sanitari Integral, L’Hospitalet de Llobregat (Barcelona)

Although numerous serological tests have been developed for TB serodiagnosis, none of these have shown adequate levels of accuracy; in consequence, they have not been widely implemented. Diverse mycobacterial antigens have been evaluated in these tests, among which are cell-wall glycolipidic antigens such as cord factor (CF). This molecule is made up of a trehalose residue sterified to two mycolic acids (MAs); it has been established that MAs are the epitopes for the anti-CF antibodies recognition. Furthermore, a possible cross-reaction between anti-MAs antibodies and cholesterol (COL) in TB patients has recently been published. The purpose of this study was to detect and compare the presence of IgG, IgM and IgA antibodies against MAs with regard to the presence of anti-CF and anti-COL antibodies in TB patients. To address this question, MAs and CF were purified from M. tuberculosis H37Rv (ATCC 27294T), and commercially available COL was used. An ELISA was developed to detect IgG, IgM and IgA antibodies to MAs and COL, and an ELISA previously developed for CF was performed. The presence of IgG, IgM and IgA anti-MAs, CF and COL in the sera of 31 HIV-negative TB patients at the time of TB diagnosis and throughout anti-TB prophylaxis, 20 PPD-positive donors, 20 PPD-negative donors and 20 patients affected by other pneumonias were determined. No antibodies to MAs were determined in any sera of the groups studied, whether for TB patients at the onset or during prophylaxis, or in control groups. Anti-CF IgG and IgA antibodies in sera from TB patients were detected, following a downward kinetic from the beginning to the end of the prophylaxis. Test sensitivity and test specificity were 41% and 78%, respectively, for IgG detection, and 19% and 95%, respectively, for IgA detection. Additionally, anti-CF IgM antibodies were detected in all the groups. In contrast, neither IgG nor IgA anti-COL antibodies were detected in any of the groups; however, IgM anti-COL antibodies were also detected in sera from all the groups studied. Each serum showing anti-CF and COL antibodies was individually analysed, and their titres against each antigen were compared. Analysis showed that response profiles were different against the two antigens. Moreover, given that the sera reacting against COL or/and CF did not react against MAs, it is therefore possible to rule out a cross-reaction between MAs and COL. The overall results invalidate MAs as possible antigens for the serodiagnosis of TB.

158 ESM 2009 PP-87

Mycobacterium tuberculosis

Arley Gómez López Infectious Diseases and Tropical Medicine Unit; Universidad del Rosario

TlyA protein has a controversial function as a virulence factor on Mycobacterium tuberculosis (Mtb). Updated studies have not demonstrated a possible hemolytic activity conferred by TlyA and contrary to recent evidence have suggested a function enzymatically similar to RNA methyltransferase at ribosomal level which confers antibiotic susceptibility to capreomycin and viomycin. Our aim was to determine the In vitro hemolytic activity of Mtb TlyA overexpressed and purified from E. coli BL21-AI and to carry out an in silico phylogenetic and structural analysis. Based on tlyA gene sequence (Rv1694) from Mtb H37Rv specific primers were designed. The amplification product was ligated on pEXP5-CT/TOPO vector (Invitrogen), transformed and overexpressed on E. coli BL-21-AI. After purification by affinity chromatography, TlyA-His6 recombinant protein was analyzed by SDS-PAGE under denaturing conditions which was detected as 28 KDa single band. Recombinant protein as recognized by anti-His monoclonal antibody. Protein structural characterization by circular dichroism was carried out. On the other hand, hemolytic activity assays using TlyA-His6 purified were negative as well as on bacterial lysates. Hemolytic assays with TlyA-His6 supplemented with calcium and magnesium were negative suggesting lack of specific requirements for this activity. By using bioinformatics tools, a ribosomal binding called S4 located between 5 and 68 residues and FtsJ among 62 and 247 residues were identified on TlyA. These domains have been described on proteins associated with ribosomal trans- lational machinery. The Hidrophobicity profile was in disagreement with a possible transmembrane helix although non polar amino acid composition suggesting that TlyA might not be membrane attachment. Nevertheless, three-dimensional model (Structural homology with 1L9K model) reveals a consensus structure with a common core, comprising a parallel β-sheet of six strands, sandwiched between two layers of α-helices corresponding to a RNA methyltransferase structure. Phylogenetic analyses showed that TlyA is highly conserved among mycobacteria species and it does not exhibit changes among Mtb complex strains. tlyA gene evolution might operate under purifying selection model. Additionally differences were observed among TlyA and bacterial pore forming proteins. This evidence supports a link between ribosomal modifications to posttranslational level and suggests a functional an- notation error of this family protein at GENBANK as well as missannotation on several genomes as Mtb genome. Studies on resistance mechanisms of Mtb mediated by ribosomal proteins might be useful for understanding new alternative therapeutic approaches for tuberculosis control applied to knowledge of second line antibiotics such as capreomycin.

Key words TlyA, Mycobacterium tuberculosis, hemolysin, Structural modeling, RNA methyltransferase.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 159 PP-88

Evaluation of the applicability of serodiagnosis for tuberculosis in Portugal

Carina Ferreira 1, Andrea Afonso 2, Raquel Duarte 3, Konstantin Lyashchenko 4, Anabela Silva 5, Filomena Rodrigues 5, Anabela Miranda 5, Margarida Tavares 6, Cátia Caldas 6, Fátima Valente 2, António Valente 2, Olga Vasconcelos 7, Joana Amado 7, Margarida Correia-Neves 1 1 - Life and Health Sciences Research Institute (ICVS), University of Minho, Braga, Portugal 2 - Centro de Saúde de Bragança, Bragança, Portugal 3 - Centro de Diagnóstico Pneumológico de Vila Nova de Gaia, Portugal 4 - Chembio Diagnostic Systems, New York, USA 5 - Centro de Tuberculose e Micobactérias, Instituto Nacional de Saúde Dr Ricardo Jorge, Delegação do Porto, Portugal 6 - Serviço de Doenças Infecciosas, Hospital de São João, Porto, Portugal 7 - Hospital Joaquim Urbano, Porto, Portugal

Diagnosis of tuberculosis requires laboratory techniques to complement clinical information. Among the different techniques in use, the only accurate diagnostic method is based on the search for Mycobacterium tuberculosis growth in cultures of human biological samples, usually sputum. However, this approach is long, lacks sensitivity for samples with low bacterial load, and is invasive in the case of non-pulmonary tuberculosis. In order to overcome these constraints, several novel methodological approaches are under evaluation. Among these is the determination of tuberculosis-specific antibodies in the sera - serodiagnostic methods. Validation of serodiagnosis tests in any given country is mandatory, since sensitivity and specificity vary depending on factors such as the composition and frequency of environmental mycobacteria, previous vaccination with BCG, prevalence of tuberculosis and other diseases, and host genetic background. We evaluated the specificity and sensitivity of two serodiagnosis tests: TB STAT-PAK II - an immunochromatographic test for the detection of antibodies to Mycobacterium tuberculosis; and MAPIA - Multi-Antigen Print Immunoassay (both developed at Chembio Diagnostics, NY, USA). The specificity of the tests was very high, ranging from 94 to 100% depending on the test and control population analysed. The sensitivity was 37 and 54% for TB STAT-PAK II and MAPIA, respectively, and it increased during the first 3 months of treatment, for TB STAT-PAK II. Interestingly, when the smear results were combined with the ones from the TB STAT-PAK II, the sensitivity increased from 71 (just smear) to 79%. Thus, taking into consideration the great specificity of the TB STAT-PAK II, its use for smear negative patients could increase the rate of detection in early TB diagnostic.

160 ESM 2009 PP-89

IMPLEMENTATION OF THE THIN LAYER AGAR (TLA) FOR THE DIAGNOSIS OF SMEAR NEGATIVE PULMONARY TUBERCULOSIS IN A HIGH HIV PREVALENCE SETTING

Martin, Anandi 1, Munga Waweru, Peter 2, Babu Okatch, Fred 2, Amondi Ouma, Naureen 2, Bonte, Laurence 2, Palomino, Juan Carlos 1, Varaine, Francis 2, Portaels, Françoise 1 1 - Institute of Tropical Medicine, Antwerp, Belgium 2 - Médecins Sans Frontières, Paris, France

Early diagnosis of smear-negative pulmonary tuberculosis in countries with high incidence of TB/HIV co-infection is crucial to limit the mortality and control the disease. The objective of this study was to evaluate the performance of a low-cost method, the Thin Layer Agar (TLA), for the diagnosis of smear-negative patients compared to the gold standard Löwenstein-Jensen method. TLA relies on microscopic detection of cording growth that is characteristic of M. tuberculosis and is able to differentiate between M. tuberculosis and non-tuberculous mycobacteria. This prospective study was performed in Homa Bay district Hospital in Kenya. Out of 1584 smear-negative sputum samples, 212 were positive by LJ (13.5%) and 220 positive by TLA (14%). The sensitivity of LJ and TLA was 71% and 74 % respectively. With further improvement for decreasing the contamination rate in both methods, TLA could become an affordable method for the diagnosis of smear-negative tuberculosis in resource-limited settings allowing the simultaneous detection and identification of M. tuberculosis, within 2 weeks.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 161 PP-90

DIRECT DETECTION OF RIFAMPIN RESISTANCE IN Mycobacterium tuberculosis BY THE NITRATE REDUCTASE ASSAY APPLIED DIRECTLY IN SPUTUM SAMPLES

Regina Ferro e Silva 1, Maria de Lourdes Shikama 1, Gleize Villela 1, Daisy Nakamura Sato 1, Carmen Maria Saraiva Giampaglia 1, Maria Conceição Martins 1, Anandi Martin 2, Juan Carlos Palomino 2 1 - Instituto Adolfo Lutz (IAL), São Paulo, Brazil 2 - Institut of Tropical Medicine, Antwerpen, Belgium

Resistance to rifampin (RIF) is an important predictor for the early diagnosis of MDRTB. Conventional methods for DST of M. tuberculosis require several weeks to give results.Thus, an alternative in vitro method which can detect drug susceptibility directly from sputum and presents quick results and low cost, will be very useful for tuberculosis diagnosis. The nitrate reductase assay uses the detection of nitrite as an indication of growth when used as a drug susceptibility test. Objective:to compare the nitrate reductase assay (NRA) with the proportion method (PM), considered as gold standard, to detect RIF resistance in M. tuberculosis directly from sputum samples. Method: the study was carried out by 4 regional laboratories from the state of São Paulo, Brazil. A total of 206 sputum samples tested smear positive from patients with pulmonary tuberculosis. The sputum was decontaminated by Petroff method and DST to RIF was carried out using the PM and the NRA. Nitrate Reductase Assay: Sputum samples, after decontamination were inoculated in one tube control (without drug) and one tube containing 40 µg/ml of rifampicin. Findings: the results of the DST obtained directly from sputum were: 6 samples resistant to RIF and 200 samples susceptible. The comparison between NRA and the traditional gold standard method showed agreement of 100%. The sensitivity and specificity of the NRA for rifampicin was 100%. Results were available in 10 days for 66 (34%) samples, 15 days for 102 (53%) samples and 20 days for 24 (13%) samples while the results of PM took 30 days to be available. Conclusions: the NRA proved to be a promising method for the screening of suspect MDRTB cases directly from sputum samples. The simplicity of the method, its low cost and celerity to give the results make it a good alternative method for laboratories in resource-poor settings.

162 ESM 2009 PP-91

DIRECT DETECTION OF RIFAMPIN RESISTANCE IN Mycobacterium tuberculosis BY THE NITRATE REDUCTASE ASSAY APPLIED DIRECTLY IN SPUTUM SAMPLES

Ferro Regina Silva 1, Shikama Maria de Lourdes 1, Villela Gleize 1, Sato Daisy Nakamura 1, Giampaglia Carmen Saraiva 1, Martins Maria Conceição 1, Martin Anandi 2,3, Palomino Juan Carlos 2, Telles Maria Alice da Silva 1 1 - Instituto Adolfo Lutz, São Paulo, Brazil 2 - Institute of Tropical Medicine, Antwerp, Belgium 3 - Médecins Sans Frontières, Paris, France

A cost-effective and rapid drug susceptibility testing (DST) method is required to guide TB treatment. Commercially available systems such as BACTEC MGIT 960 and MB/BacT are faster but demand costly equipment and supplies, therefore, are not feasible in most resource-poor settings. Resistance to rifampin (RIF) is an important predictor for the early diagnosis of MDRTB. To compare the nitrate reductase assay (NRA) with the proportion method (PM) to detect RIF resistance in M. tuberculosis directly from sputum samples. The study was carried out by 4 regional laboratories from the state of São Paulo, Brazil. A total of 210 sputum samples tested smear positive from patients with pulmonary tuberculosis. The sputum was decontaminated by Petroff method and DST to RIF was carried out using the PM and the NRA. The results of the DST obtained directly from sputum were: 6 samples resistant to RIF and 204 samples susceptible. No discordance was observed between the two methods. The sensitivity and specificity of the NRA was 100%. Results were available in 10 days for 75 (36%) samples, 15 days for 107 (51%) samples and 20 days for 28 (13%) samples. The results of PM took 30 days to be available. The NRA proved to be a promising method for the screening of suspect MDRTB cases directly from sputum samples. The simplicity of the method, its low cost and celerity to give the results make it a good alternative method for laboratories in resource-poor settings.

Acknowledgements This study was partially funded by INCO-Dev ICA4-CT-2001-10087.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 163 PP-92

MTBDRPLUS ASSAY IS A USEFUL TOOL TO SCREEN FOR MULTI-DRUG RESISTANT TUBERCULOSIS IN A NATIONAL SURVEY

De Haas, Petra 1, Ramona Zenhorst 2, Pike Mwamba 2, Mweemba Muvwimi 3, Winnie Mwanza 2, Grace Mbulo 2, Nathan Kapata 2, Helen Ayles 1 1 - Zambart project, Lusaka, Zambia and LSHTM London 2 - Zambart project, Lusaka, Zambia 3 - National Reference laboratory, Lusaka, Zambia

Background The World Health Organization requests that countries conduct tuberculosis drug resistance surveys (DRS) every five years to monitor trends of drug resistance and to determine rates of multi-drug resistant tuberculosis (MDR-TB). Zambia conducted its second nation-wide DRS in 2008. The objective of this study is to investigate whether the MTBDRplus assay (HAIN), a new molecular assay performed directly on sputum, is a useful tool in conducting a DRS.

Method Throughout Zambia approximately 900 sputum specimens were collected from consecutive smear-positive TB patients and transported to the TB Reference Laboratory in Lusaka. Specimens were decontaminated and concentrated smears were prepared. MGIT and LJ cultures were inoculated. Drug susceptibility testing (DST) was performed on positive cultures. Remaining decontaminated sputum was heat-killed, sonicated and stored at -80C. The MTBDRplus assay was performed using a 1:5 or 1:10 dilution of decontaminated sputum. Results: Of the first 340 specimens tested using the MTBDRplus assay, 307 (90.3%) showed no evidence of resistance, while thirty-three (9.7%) showed mutations consistent with resistance: 10 were MDR-TB, 20 were isoniazid (INH) mono-resistant and 3 were rifampicin (RIF) mono-resistant. MGIT DST results were obtained from 271 (79.7%) of 340 specimens with an MTBDRplus result. We were unable to obtain MGIT DST results from the remaining 69 (20.3%) specimens due to contamination or lack of growth. Thirteen (39.4%) of 33 specimens that showed mutations consistent with resistance in the MTBDRplus assay failed to yield a DST result on MGIT. Six out of these 13 samples are according to the MTBDRplus assay MDR, 5 are INH mono-resistant and 2 RIF mono-resistant . One sample that showed RIF mono- resistance using the MTBDRplus assay, showed both isoniazid and rifampicin resistance uses the MGIT DST. Conclusion In our study, the MTBDRplus assay performed directly on sputum was more rapid and cost-effective than culture to screen for MDR-TB.

164 ESM 2009 PP-93

CONTRIBUTION OF LABORATORY FACTORS TO HIGH MGIT CULTURE CONTAMINATION RATE IN ZAMBIA de Haas, Petra 1, Moyoyeta, Monde 2, Samutela, Mulemba 2, Mwanza, Winnie 2, Musunsa, Alan 3, Mbulo, Grace 2, Muvwimi, Mweemba 3, Ayles, Helen 1 1. Zambart project, Lusaka, Zambia and LSHTM, London 2. Zambart project, Lusaka, Zambia 3. National reference laboratory, Lusaka, Zambia

Background: An evaluation study of the MGIT liquid culture system in Zambia found a high contamination rate. It was suggested that factors such as delayed sample processing due to long distances, inadequate storage of samples once submitted, poor laboratory infrastructure and inexperienced staff may have contributed to the high contamination. The objective of this study was to investigate the contribution of laboratory factors to the higher than expected rate of MGIT contamination.

Method: As part of the National drug resistance survey, 917 sputum specimens were collected from smear-positive TB patients and transported to the TB Reference Laboratory in Lusaka. After decontamination using NALC-NAOH (1,5% final concentration) MGIT and LJ cultures were inoculated. In addition 584 of the decontaminated samples were inoculated onto blood agar plates (BA). When the MGIT culture showed growth Ziehl Nielsen staining was performed. If micro-organisms other then acid fast bacilli were seen, a BA was inoculated and subsequent colonies identified using biochemical tools.

Results Time between sample submission and processing varied from 1-50 days with a median of 9 days. Increased contamination in MGIT was found when the time between sample submission and processing was extended; 18.6% for 1-7 days, 27.4% for 8-14 days and 37.5% for more than 15 days. The overall contamination rate was 27.6% (95% CI 24.0-31.4). Of 584 decontaminated sputum 197 (33.7%) showed growth on BA. Out of the 197 samples showing growth 86 (43.6%) were also contaminated in MGIT whereas for samples that showed no growth on BA, 70 (18%) out of 387 were contaminated. Contaminated organism from the sputum and from the contaminated MGIT culture were identified for 35 out of the 86. Identical species were identified in only sixteen (45.7%) of these 35 samples, whereas in 19 (54.3%) cases a different contaminanting species was found in the MGIT culture compared to the decontaminated sample.

Conclusion High contamination on MGIT is a problem in our setting. Delayed processing of samples increases the chance of samples being contaminated. The contamination of samples that did not show any growth on BA from decontaminated sputum as well as different species isolated from the contaminated MGIT compared to the decontaminated sputum suggest that a major part of the contamination may be due to laboratory factors.

Oral Communication

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 165 PP-94

PROGRAMMATIC COMMUNITY-BASED MANAGEMENT OF MDR-TB: EXPERIENCE IN KARACHI, PAKISTAN

Ahmed, Altaf; Qazi, Fahad; Khan, Amir Javed The Indus Hospital, Karachi, Pakistan

Introduction Multidrug Resistant Tuberculosis (MDR-TB) is defined as TB resistant to the two most powerful anti-TB drugs, Isoniazid (H) and Rifampicin (R). Pakistan is ranked 8th among the high TB burden countries.In November 2007, the Karachi DOTS-Plus program was established with an objective to provide free, comprehensive, community-based management of MDR-TB patients in Karachi and Hyderabad based on Partners in Health (PIH) and World Health Organization (WHO) guidelines. The purpose of this paper is to share our initial experience at programmatic management of MDR-TB in a low income setting.

Methodology At baseline registration, smears, cultures, DST, radiology, and ancillary tests were performed on each patient and each patient house was mapped using GPS devices. Inclusion criteria were as follows: 1) Acid Fast Bacilli Culture and Sensitivity (AFBCS) showing MDR-TB or PDR-TB 2) Clinical, Radiological or Bacteriological (smear and culture) evidence of active disease Enrollment was based on availability of funds, clinical judgment and proximity to center. All patients were managed in the community. Each enrolled patient received monthly consultation, uninterrupted and monitored second line drug supply, laboratory tests as per program guidelines. Social support was also provided to all patients in the form of: 1) Monthly Food Baskets 2) Professional Counseling 3) Treatment Supporters (TS)

Results As of May 2009, 106 MDR-TB patients were registered in the program (49 males, 57 females). The mean age was 29.58 (+/-12.8). 73 patients were put on treatment. Their classification according to previous treatments was as follows: 1) After failure of retreatment ……30 (41%) 2) Relapse ………………………...14 (19%) 3) After failure of first treatment …12 (16%) 4) New …………………………….6 (8%) 5) Registered after default…………5 (7%) 6) Other…………………………….1 (1%) 7) Transfer in ………………………1 (1%)

68 (90%) patients had received first line treatment previously, 1 (2%) had received second line treatment and 6 (8%) were new patients. HIV testing on 68 of 73 enrolled patients came negative for all. Adverse events recorded were recorded. Comorbid conditions included DM, Tobacco use, substance abuse, chronic lung disease, hepatitis, respiratory failure, preg- nancy, hypertension, gastritis, renal failure and neuropathy. Although treatment duration for the first pool of p

166 ESM 2009 PP-95

EVALUATION OF CAPILIA (TAUNS) FOR RAPID IDENTIFICATION OF MYCOBACTERIUM TUBERCULOSIS COMPLEX FROM CULTURES

C Muchwa2,3, J Akol2,3, F Mumbowa5, P Orikiriza2,3, K Morgan2,3, K Eisenach4 , M Joloba1,3, A Etwom2,3, P Mugyenyi2, R Mugerwa3 1 - Department of Medical Microbiology, Makerere University Medical School, Kampala, Uganda 2 - Joint Clinical Research Centre, Kampala, Uganda 3 - Uganda-CASE Research Collaboration, Kampala, Uganda 4 - University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, USA;. 5Infectious Diseases Institute (IDI), Kampala, Uganda

Introduction The Capilia TB assay is a simple immunochromatographic assay which uses anti-MPB64 monoclonal antibodies to discriminate MTB from non-tuberculosis mycobacteria. Evaluation of Capilia to determine its costs, performance and turn around time (TAT) was done using PCR IS6110 assay (PCR) as a gold standard.

Methods Respiratory and blood samples specimens were digested and decontaminated using 1.5% NAOH/NALC, concentrated by centrifugation and inoculated into BACTEC MGIT 960 culture tubes for incubation. Blood was aseptically inoculated and incubated in the BACTEC 9120 instrument. All BACTEC positive cultures were screened for acid fast bacilli by the Ziehl-Neelsen method before testing for MTB. Blood cultures were then inoculated on 7H10 agar and incubated for MTB isolation. The Capilia test was performed according to the manufacturer’s instructions while PCR was done according to laboratory protocol. The test included 155 respiratory and 70 blood samples were tested.

Results Overall agreement between Capilia and PCR IS6110 methods was 98.2%. Capilia achieved a sensitivity of 98.4% and specificity of 97.9%. Initial PCR comparison for respiratory cultures resulted in sensitivity and specificity of 97.4% and 98.7% respectively. Blood achieved specificity of 27.4% only; this may be due to false negative PCR results caused by PCR inhibitors in blood cultures. PCR testing was performed from colonial growth on 7H10 accuracy and reliability of PCR as a gold standard and the resulting Capilia test sensitivity increased to 100% and 94.4% specificity.

Conclusion The Capilia has an overall sensitivity of 98.4% and specificity of 97.9% and is far more accurate method of identifying MTB directly in blood cultures. It is less expensive (≈ $5) compared to PCR (≈ $45). It is easier to perform with TAT of 20 minutes while PCR has TAT of 8 hours for respiratory cultures.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 167 PP-96

COMPARISON OF CAPILIA (TAUNS) AND IS6110 PCR FOR RAPID IDENTIFICATION OF Mycobacterium tuberculosis COMPLEX FROM CULTURES IN KAMPALA, UGANDA.

C Muchwa2,3, J Akol2,3, P Orikiriza2,3, K Morgan2,3, F Mumbowa5, K Eisenach4, A Etwom2,3, M Joloba13 1 - Department of Medical Microbiology, Makerere University Medical School, Kampala, Uganda 2 - Joint Clinical Research Centre, Kampala, Uganda 3 - Uganda-CASE Research Collaboration, Kampala, Uganda 4 - University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, USA 5 - Infectious Diseases Institute (IDI), Kampala, Uganda

Introduction The Capilia TB assay, a simple immunochromatographic method which uses anti-MPB64 monoclonal antibodies to discriminate MTB from non-tuberculous mycobacteria has been successfully evaluated in other settings. Before adopting capilia in our laboratory, an evaluation to determine its costs, performance and turn around time (TAT) compared to the existing molecular identification method using PCR IS6110 assay (PCR) was done.

Methods Sputum/Gastric samples were processed using the 1.5%NAOH/NALC decontamination method and the sediment cultured using the BACTEC MGIT 960 system.. Blood/Pleural fluids were aseptically inoculated in the BACTEC 9120 instrument. All BACTEC positive cultures, (sputum and body fluids) were screened for acid fast bacilli by the Ziehl- Neelsen method before testing for MTB. The Capilia test was performed on all ZN positive MGIT 960 tubes according to the manufacturer’s (TAUNS ) instructions while the PCR was done according to laboratory protocol. The ZN positive

blood and other body fluid cultures were inoculated on 7H10 agar for MTB isolation. Capillia was performed on ZN positive isolates.The test included 155 sputum/gastrics and 70 body fluid samples. The Capilia and PCR assays were done by separate technicians who were blinded to the other test results.

Results There was an overall agreement of 98.2% between the Capilia and PCR IS6110 methods. Capilia achieved a sensitivity of 98.4% and specificity of 97.9%. These results concurred with the finding of Jann Wang et al publication of 98.6% & 97.9% respectively. The initial PCR comparison for sputum/gastric cultures resulted in a 97.4% and 98.7% sensitivity and specificity respectively. When the PCR test was performed from colonial growth on 7H10, the accuracy and reliability of PCR as a gold standard increased and the resulting Capilia test sensitivity was 100% and 94.4% specificity. The sensitivity and specificity of Capilia on contaminated cultures was 97.0% and 98.7% respectively while that on pure cultures was 99.0% and 94.4%. There was no significant difference in the test performance between contaminated and pure cultures (p values of 0.12405 and 0.786 respectively). The recurrent cost for capilia was ~$5 and that of PCR ~$45. The average TAT for Capilia was 20 minutes while that of PCR 8 hours for sputum/gastric cultures and 22 days for blood cultures. Overall Capilia was much easier to learn, had fewer steps and requires no instruments

Conclusion/Discussion • The sensitivity and specificity of Capilia is comparable to PCR for both pure and contaminated MTB cultures. • Capilia is less expensive, faster and easier to perform than PCR. • Capilia is capable of identifying MTB directly in blood cultures.

168 ESM 2009 PP-97

DIRECT TESTING FOR MULTI DRUG RESISTANT TUBERCULOSIS WITH FOUR ASSAYS EVALUATED AT KAMPALA, UGANDA

Freddie Bwanga1,2,3, Sven Hoffner2,3, Melles Haile2,3, Moses L Joloba1 1 - Department of Medical Microbiology Makerere University College of Health Sciences Kampala, Uganda 2 - Department of Bacteriology, Swedish Institute for Infectious Diseases Control, Solna Sweden 3 - Department of Microbiology, Tumour and Cell Biology (MTC), Karolinska Institute, Stockholm, Sweden

Background Multi drug resistant tuberculosis (MDR TB) is on the rise worldwide. Early detection of MDR TB is important for effective control of MDR TB transmission. In most TB high burden countries this remains a challenge due to lack of rapid tests. This study evaluated four rapid tests for MDR TB detection.

Methods Smear positive re-treatment TB patients were consecutively recruited at Mulago – Uganda’s National referral Hospital. Samples were processed using a final concentration of 1.5% NAOH-NALC method. Sediments were used directly to set susceptibility to isoniazid and rifampicin with four rapid tests at the National TB Reference Laboratory Kampala. The four tests included the Nitrate Reductase Assay (NRA), Microscopic Observation Drug Susceptibility (MODS), Mycobacterium Growth Indicator Tube (MGIT 960) and Genotype® MTBDRplus (Hain Life Sciences, Nehren, Germany). Results of the four tests were compared to those of the conventional indirect Lowenstein-Jensen proportion method (L-J PM).

Results A total of 66 patients were recruited. Interpretable results were obtained for all the samples with the LJ PM and MODS assay, 64 (97%) with the NRA and MGIT 960, and 62 (94%) with the Genotype® MTBDRplus. Interpretable results across all the five tests were available for 56 samples and results obtained on initial testing were 100%, 98%, 91%, 82% and 68% with the Genotype® MTBDRplus, MODS, LJ PM, NRA, and MGIT 960, respectively. Repeat testing with the MGIT 960 was due to power failure -13 samples, contamination - 4 samples and undergrowth - one sample. Sensitivity and specificity for detection of resistance to isoniazid was 100% and 100% for NRA, 100% and 95% for MODS, 93% and 98% with the Genotype® MTBDRplus, and 93% and 100% with the MGIT 960, respectively. For rifampicin it was 100% and 100% with NRA, 91% and 93% with MODS, 100% and 96% with Genotype MTBDR®plus, and 73% and 100% with the MGIT 960, respectively. The average time to results was 2 days (range 1-3 days) for Genotype® MTBDRplus, 8 days for MGIT 960 (range 5-13 days), 8 days for MODS (range 7-18 days) and 11 days for NRA (range 10-21 days). Results obtained within 10 days were 91%, 88%, and 75% for the MODS, MGIT 960, and NRA, respectively.

Conclusion Findings show excellent performance of the direct NRA, MODS, and Genotype® MTBDRplus for MDR TB detection, with most of the interpretable results obtained on initial testing in <14 day.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 169 PP-98

PEA PRODUCTION BY MYCOBACTERIA AND ITS APPLICATION IN A RAPID DRUG SUSCEPTIBILITY TEST.

McNerney, Ruth 1, Turner, Claire 2, Mallard, Kim 1, O’Sullivan, Denise 1 1 - London School of Hygiene & Tropical Medicine 2 - Cranfield University

Metabolites produced during bacterial growth may be used to monitor the impact of drugs on bacteria. We have investigated volatile organic compounds emitted by mycobacteria growing on Lowenstein Jensen (LJ). Mass spectrometry determined one of the major volatile compounds from M. bovis BCG to be phenylethyl alcohol (PEA), a bacteriostatic compound that is a reversible inhibitor of DNA synthesis. PEA production was also observed in mycobacteria other than tuberculosis (MOTT). That such a compound is produced in quantity by mycobacteria growing on LJ is surprising and may explain the limited growth of mycobacteria on this media. PEA is only produced during bacterial growth and monitoring production during exposure may provide a means of determining susceptibility to antimicrobial compounds. To test this hypothesis, bacteria were placed on LJ slopes containing drug and incubated at 37C. Headspace vapors from the culture tubes were analysed using the zNose, an ultra-rapid capillary gas chromatograph coupled to a SAW detector. The test required no sample preparation and each slope was tested in less than 2 minutes. To minimize the risk of creating infected aerosols culture tube caps incorporated a PTFE/silicone septum enabling air to be drawn from the tube without remov- ing the cap. Samples collected were heat sterilized during testing. Headspace samples from drug containing slopes were compared to control slopes without drug. The incubation time required for differentiation between positive and negative samples or ‘growth’ and ‘no growth’ depended on the size of the inoculum and the species of mycobacteria and ranged from hours to a few days. MOTT could be differentiated from M. tuberculosis complex bacilli by continued production of PEA in the presence of 500ug/ml para-nitrobenozic acid. We conclude that detection of volatile metabolites emitted by mycobacteria growing on Lowenstein Jensen offers a simple, rapid alternative to assess their drug susceptibility.

170 ESM 2009 PP-99

VARIOUS STRATEGIES TO DECONTAMINATE ACID FAST BACILLI POSITIVE LIQUID CULTURES FROM BACTEC MGIT 960

Balmoi, Faith Joint Clinical Research Centre, Kampala, Uganda

Introduction Though liquid culture is enriched and more sensitive in the recovery of MTB, it yields a greater percentage of contaminants as well. It is essential that MTB cultures be isolated pure to permit drug susceptibility testing which is vital for prognosis of patients besides determining the presence of MDR-TB and XDR-TB. In this study we evaluated various approaches to reduce or eliminate contamination in Bactec MGIT 960 cultures.

Method Contaminated AFB PCR positive MGIT cultures were subjected to decontamination methods of: i) Double PANTA Polymyxin B (750µg/ml); Amphotericin (75µg/ml); Nalidixixic (300µg/ml); Trimethoprim (75µg/ml) and Azlocillin (75µg/ml) ii) VAN vancomycin (30.5µg/ml), Amphotericin (106µg/ml) and Nalidixic acid (226.7µg/ml) iii) 2% NaOH each contaminated culture was divided and subjected to all three decontaminating procedures. The decontaminated cultures were then inoculated in MGIT tubes and 7H10 selective whole plates and incubated in Bactec MGIT 960 and a 5%-10% CO2 at 37°C respectively. The plates were read weekly until a sufficient colonial growth was achieved. The positive MGIT was subjected to Ziehl- Neelsen (ZN) smear and blood agar culture for purity check.

Results A total of 50 specimens had analyzable results for each method. Double PANTA was able to recover 62.0%, VAN 60.0% and 2% NaOH 24.4% while 16.0%, 14.0% and 6.7% respectively remained contaminated. However, 22.0%, 26.0% and 68.9% were non-viable after decontamination with double PANTA, VAN and 2% NaOH respectively. Further testing is being done so as to generate more reliable results.

Conclusion • VAN and double PANTA were comparable as far as giving more specimens (p value = 0.614). With pure cultures when they are used for decontamination. • More specimens were unrecoverable when 2% NaOH was used than the other two methods. • The cost of decontaminating a sample using VAN was ~$1 while double PANTA was ~$5.70

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 171 PP-100

LOW COST ISOLATION OF Mycobacterium tuberculosis (MTB) FROM BLOOD

Orikiriza, Patrick Uganda-CASE Research Collaboration, Kampala

Introduction Routinely, identification of MTB from body fluids requires a PCR based technique which has been adopted as a gold standard. This technique however has been unsuccessful against blood cultures which are often affected by inhibitors. In this study, we evaluated a simple but effective way of isolating and identifying MTB in blood cultures.

Method A total of 91 blood cultures positive for acid fast bacilli were sub-cultured onto 7H10 medium and Lowenstein Jensen slants and incubated at 37ºc in a 5% carbon dioxide incubator. The isolates that grew on solid media were further tested by PCR to confirm MTB. The original blood cultures were tested by diluting an aliquot of blood culture in PCR waters and repeating the IS6110 PCR and also by Capilia for identification of MTB.

Results The study found 15 cultures PCR positive when diluted in PCR water and 56 positive with Capilia. On the other hand, cultures grown on the solid media before performing PCR yielded 53 PCR positives from 7H10, 44 PCR positives from LJ and 28 did not grow on either medium. The time it took for the cultures to turn positive on the different media was also noted

Conclusion Identification of MTB by PCR was more efficient blood cultures were sub cultured to solid media for identification assay Capilia tests performed directly from blood culture media gave comparable results (95%) with MTB identification by PCR from solid growth. There was an overall better turn around time using the 7H10 media compared to LJ medium

172 ESM 2009 PP-101

COMPARISON OF RAPID COLORIMETRIC METHOD, PROPORTION METHOD AND BACTEC460 TB SYSTEM FOR TESTING . SUSCEPTIBILITY OF M.tuberculosis TO RIFAMPINE AND ISONIASIDE

. . o Begum KAYAR, Ülkü ORAL ZEYTINLI, Ayse, KARACALI, Ayben SOYAL, Togrul Nagiyev, Fatih KÖKSAL Cukurova University, Medical Faculity

Introduction Multidrug-resistant (MDR) M. tuberculosis (MTB) strains founded resistant to at least isoniaside [INH] and rifampin [RIF], the two most important first-line drugs pose a serious threat to the control of tuberculosis (TB) and the spread of these strains has become a major public health problem. In this study, the performance of antimycobacterial susceptibility testing for the first line drugs (RIF and INH) with colorimetric nitrate reductase-based antibiotic susceptibility (CONRAS) and conventional proportion method depend on solid agar culture (LJ) were compared to BACTEC 460 TB system as the gold standard.

Methods Of the total of 187 MTB strains, when 24 isolates were found as resistant to RIF and INH by Bactec 460 TB other 163 strains were susceptible for these antibiotics. All of strains were isolated in sputum from patient with pulmonary disease and identified as MTB by NAP test in Bactec 460 system. The CONRAS test was performed with 0.1 mg/mL of INH and 1 mg/mL of RIF on LJ as modification of standart methods described by H.Syre et al . Antibiotic susceptibility testing with proportion method was performed on LJ medium according to the standard protocol laid down by WHO. Results: The sensitivity, specificity and overall agreement of the CONRAS test were 95.83% (23/24), 99.38%(162/163) and 97.60% for RIF and 83.33% (20/24), 99.38% (162/163) and 91.35% for INH, respectively. Results for proportion tests were found as; 91.66% (22/24), 93.25% (152/163) and 92.45% for RIF and 75% (18/24), 97.54% (159/163) and 86.27% for INH, respectively by Bactec 460TB system as the gold standart.

Interpretation & conclusion CONRAS test showed good agreement with Bactec 460 (the overall agreement of this test was 94.47%) for each of the antimicrobial tested. CONRAS test is simple, easy to perform, less expensive, reliable and may be used as a preliminary screen for susceptibility testing of MTB in particularly for resource-poor countries.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 173 PP-102

EVALUATION OF THE MGIT TBc ID TEST VS TWO COMMERCIALLY AVAILABLE RAPID IMMUNOASSAYS FOR M. tuberculosis COMPLEX ORGANISM DETECTION FROM LIQUID AND SOLID CULTURE

Crews, Virginia 1, Warns, Matthew 1, Pfeltz, Richard 1, Beaty, P. Shawn 1, Rosales, Julie 1, Kopher, Ken 1, Joshi, Sudhaunshu 1, Hoosen, Anwar 2, Said, Halima 2 1 - BD Diagnostic Systems, Sparks, Maryland, United States of America 2 - Department of Medical Microbiology, Faculty of Health Sciences, University of Pretoria and National Health Laboratory Service, Tshwane Academic Division, Pretoria, South Africa

Introduction Chromatographic lateral flow immunoassays for the MPT64 protein antigen can provide a rapid and cost-effective method to qualitatively detect Mycobacterium tuberculosis complex (Mtbc) organisms from acid-fast bacillus (AFB) positive cultures. The purpose of this study was to compare the sensitivity and specificity of three such devices with mycobacteria from liquid and solid media cultures.

Methods Positive liquid (BACTEC MGIT 960 7 mL tubes) and solid (BBL Löwenstein-Jensen slants) culture media seed- ed with mycobacteria provided inocula for side-by-side evaluation of two commercially available tests, Capilia TB (“Capilia”, Tauns Laboratories, Numazu, Japan) and SD Bioline TB Ag MPT64 Rapid Test (“Bioline”, Standard Diagnostics, Inc, Kyonggi-do, Korea), and the BD MGIT TBc Identification Test (“TBc ID”, BD Diagnostic Systems, Sparks, MD, USA). The MGIT TBc ID test is currently in clinical trials. Devices were inoculated per manufacturer’s instructions and visually assessed as positive for detection (visible test line) and reagent function (visible control line). RESULTS. All three devices detected each of the 20 Mtbc organisms tested (100% sensitivity), which included 15 M. tuberculosis strains. Specificities for the 18 non-tuberculous mycobacteria (NTM) tested were as follows: 100% for the TBc ID device, 94% for the Capilia device (due to cross-reactivity with M. marinum), and 94% for the Bioline device (due to cross-reactivity with M. gastri). Additional testing with the two cross-reactive NTM species confirmed these results. Sensitivity and specificity results for a given device were identical between solid and liquid media. However, flow issues after inoculation from solid medium as well as background discoloration after inoculation from liquid medium occurred regularly with the Bioline device.

Conclusions The three rapid Mtbc organism identification immunoassay products exhibited very good sensitivity. The specificity demonstrated by the TBc ID device was also very good, but specificity was lower for the Capilia and Bioline devices due to cross-reactivity with specific NTMs.

174 ESM 2009 PP-103

NITRATE REDUCTASE ASSAY APPLIED TO DIRECT DETECTION OF DRUG RESISTANCE IN MYCOBACTERIUM TUBERCULOSIS.

Montoro, Ernesto 1, Milián, Yoslaine 1, Lemus, Dihadenys 1, Echemendía, Miguel 1, Yzquierdo, Sergio 1, Martin, Anandi 2, Van der Stuyft, Patrick 2, Palomino, Juan Carlos 2 1 - Institute of Tropical Medicine Pedro Kourí (IPK), Havana, Cuba 2 - Institute of Tropical Medicine, Antwerp, Belgium

Tuberculosis still represents a major public health problem; especially in low-resource countries were the burden of the disease is more important. Standard methods for drug susceptibility testing of Mycobacterium tuberculosis, such as the proportional method (PM), the absolute concentration method, and the resistance ratio method, are used globally but depend on culture on solid media and are therefore time-consuming. The time lag is a significant threat to the patient, the community, and health care workers. To reduce this period, we have evaluated the nitrate reductase assay (NRA) on smear-positive sputa for the direct detection of drugs resistance in Mycobacterium tuberculosis. A total of 63 smear- positive sputum were used in this study. The samples were decontaminated using the modified Petroff method, a por- tion was used to carried out the NRA as susceptibility test to isoniazid (INH, 0,2 µg/mL), streptomycin (SM, 4 µg/mL), ethambutol (EMB, 2 µg/mL) and rifampicin (RIF, 40 µg/mL). The resulting sample was used to perform the indirect PM on Löwenstein-Jensen which was used as gold standard method. The NRA results were obtained between 14 and 28 days. However, were necessary 28 or 42 days to obtain PM results. The sensitivity of MNR to INH, SM, EMB and RIF was 88,9%, 75%, 0% and 71,4% respectively. The low sensitivity obtained for all drug evaluated was due to a small amount of resistance strains founded. On the other hand, the specificity of MNR to each drugs was 96,3% (INH), 96,1% (SM), 95,2% (EMB) and 98,2% (RIF). In general, the concordance between MNR and PM was 95,2% to INH, 92,1% to SM, 93,7 to EMB and 95,2% to RMP. In conclusion, the MNR applied in sputum samples is more rapid than any conventional method. Two drugs that define multidrug-resistance and are the most potent in the treatment of tuberculosis, INH and RIF, showed the higher values of concordance wit PM. The MNR results are easy to performance, use each drug with identical critical concentration in the same solid media than PM and could be a useful tool for detection of tuberculosis drug resistance in low-resource countries with limited laboratory facilities.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 175 PP-104

USE OF NICOTINAMIDE IN COLORIMETRIC METHODS FOR RAPID DETECTION OF PYRAZINAMIDE RESISTANCE IN MYCOBACTERIUM TUBERCULOSIS

Montoro, Ernesto 1, Lemus, Dihadenys 1, Madruga, Mariela 1, Mirabal, Niuris 1, Milián Yoslaine 1, Yzquierdo, Sergio 1, Echemendía, Miguel 1, Martín, Anandi 2, Van der Stuyft, Patrick 2, Palomino, Juan Carlos 2 1 - Institute of Tropical Medicine Pedro Kourí (IPK), Havana, Cuba 2 - Institute of Tropical Medicine, Antwerp, Belgium

Pyrazinamide (PZA) is one of the most effective anti-tuberculosis drugs. It is also bactericidal to semidormant Mycobacterium tuberculosis and it reduces the total treatment time. The current susceptibility testing methods for this drug are difficult due to the poor growth of the bacteria in acid medium which is required for drug activity. One alternative has been the use of nicotinamide (NIC), an analogue of PZA that can be converted by PZAase into active form in a physiological pH that does not hinder bacterial growth. Recently, the NIC has been applied successfully in inexpensive susceptibility testing such as nitrate reductase assay (NRA) for rapid detection of PZA resistance. The purpose of this study was to develop the NRA and malachite green indicator (MGI), both with NIC, as rapid susceptibility testing to PZA in M. tuberculosis. The NRA and MGI were carried out on 120 M. tuberculosis strains from the collection at Tuberculosis National Reference Laboratory. The concentration of NIC applied in both methods was 1 000 µg/mL and all results were compared with Wayne method which was used as gold standard, employing 100 µg/mL of PZA. The Wayne method results were obtained in 4-7 days whereas MGI and NRA results required 7-14 days. A total of 17 and 85 strains were reported as resistant and susceptible respectively by the three methods but MGI had 16 discordant results and NRA only 4 discrepancies. The MGM showed a sensitivity and specificity of 80,95% and 87,88% respectively whereas NRA provided a sensitivity of 90,48% and specificity of 97,98%. In general, the concordance of MGI and NRA were 86,67% and 96,67% respectively. The MGI employing NIC showed a low sensitivity to detect resistance to PZA. In contrast, the NRA showed a high concordance with Wayne method. This assay is rapid, accurate and could be an attractive option for rapid detection of PZA resistance, especially in limited-resource countries with high levels of resistance.

176 ESM 2009 PP-105

Implementation of liquid culture for tuberculosis diagnosis in a remote setting: lessons learned

Pamela Hepple1, Jonathan Novoa-Cain2, Chris Cheruiyot2, Elvira Richter3, Koert Ritmeijer4 1 - Manson Unit, Médecins sans Frontières, London, UK 2 - Médecins sans Frontières OCA South Sudan, Lokichoggio, Kenya 3 - Forschungszentrum Borstel, Nationales Referenzzentrum für Mykobakterien, D-23845 Borstel, Germany 4 - Médecins sans Frontières OCA, Amsterdam, the Netherlands

Issues The diagnosis of tuberculosis (TB) in Médecins sans Frontières (MSF) projects is based on sputum smear microscopy, which has low sensitivity. Following WHO recommendations, MSF established a TB liquid culture laboratory in Lokichoggio, Kenya, processing samples from 4 South Sudan projects, for diagnosis of smear-negative and extra-pulmonary (EP) TB and follow-up of patients.

Description The manual MGIT (mycobacterial growth indicator tube, Becton Dickinson) system was used with Lowenstein-Jensen media. One positive culture per patient was sent to Borstel Supranational Reference Laboratory, Germany for speciation using the Hain Genotype Mycobacteria series and sequencing techniques. From March 2007 to December 2008, sputum culture was performed for 64 diagnostic and 24 follow-up patients. Ten EP samples were also cultured. For diagnostic patients, of two smear-positives, one was culture-positive for both Mycobacterium tuberculosis (MTB) and M. fortuitum, and one for M. fortuitum. Eight of 62 (13%) smear-negatives were culture-positive for MTB complex (3 for MTB and 5 for MTBC) with nine (14%) culture-positive for other identified mycobacteria; six (9%) grew unknown, not validly-described mycobacteria, and 39 (61%) were culture negative. For follow-up patients, of seven smear-positives, one was culture-positive for MTB, one for M. intracellulare and one for M. fortuitum complex, and four (57%) were culture-negative. Among the smear-negatives, eight of 17 (47%) were culture- positive, four of which were unknown mycobacterial species, and four non-tuberculous mycobacteria (NTM). Nine (53%) were culture-negative. Samples from three EP patients of 10 grew mycobacteria, species unidentifiable. In total, only 10 of 36 (28%) culture-positive patients grew mycobacteria from sputum which could be identified as MTB or MTBC.

Lessons learned Due to the long turn-around time between sample production and species identification due to shipment issues (approximately 4-6 weeks), clinicians often did not wait for results before initiating or adjusting therapy. The high proportion of NTM was difficult to interpret. The culture results had little clinical impact, and culture lab activities were suspended in February 2009.

Recommendations Future TB culture programmes require facilities for on-site speciation of mycobacteria or, if working with reference labs, should minimize turn-around time to results by ensuring timely shipment of samples.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 177 PP-106

RAPID DETECTION OF RESPIRATORY ACTIVE MYCOBACTERIA BY AURAMINE O-CTC DOUBLE STAINING

Ichijo, Tomoaki; Izumi, Yoko; Yamaguchi, Nobuyasu; Nasu, Masao Osaka University

Introduction Clarifying the dynamics of nontuberculosis mycobacterial (NTM) and determining their hot spots in aquatic environments are important to prevent their infection to human beings. For this purpose, methods for the detection of active mycobacterial cells are required. Culture methods are widely used for detection of mycobacterial cells, but these methods are rather difficult to detect mycobacteria quantitatively and rapidly. Mycobacterial cells are quantifiable under a fluorescent microscope within several hours with acid-fast staining. In addition, several fluorochromes are available to determine the bacterial activities. In this study, we attempted to detect mycobacteria with physiological activity rapidly by combining Auramine O acid-fast staining with 5-cyano-2, 3-ditoryl tetrazolium (CTC) as an indicator of respiratory activity (Auramine O-CTC).

Methods Mycobacterium smegmatis was stained with CTC and fixed with formaldehyde. Fixed cells were stained with Auramine O and observed under an epifluorescent microscope with blue excitation.

Results And Discussion We firstly optimized the condition of the Auramine O-CTC double staining method to address the following problems; (i) CTC formazan dissolved in the decolorization step and (ii) fluorescent intensity of Auramine O was decreased by fluorescent resonant energy transfer (FRET). Specificity of this method was confirmed by staining non-targeted bacteria. Results can be obtained within 90 min by the optimized procedure, while more than one week is required for the culture-dependent approach. In conclusion, the Auramine O-CTC double staining method was useful for rapid detection of respiratory active mycobacteria. This method may contribute to identifying dynamics of NTM in aquatic environments.

Acknowledgement This work was supported by the JSPS Grant-in-Aid for Scientific Research (A)(20249007).

178 ESM 2009 PP-107

SYNERGISTIC ACTIVITY OF TWO ANTITUBERCULOUS DRUG COMBINATIONS AGAINST CLINICAL ISOLATES OF Mycobacterium tuberculosis RESISTANT TO ISONIAZID.

Emma Rey, Griselda Tudó and Julià González-Martín Servei de Microbiologia. Hospital Clínic-IDIBAPS. Dept. Microbiologia i Parasitologia Sanitaria, Universitat de Barcelona. Spanish Network for the Research in Infectius Diseases (REIPI, RD06/0008).

Objective To determine the synergistic activity of 2 drug combinations: isoniazid (H)+rifampicin (R)+ethambutol (E) and ofloxacin (O)+R against M. tuberculosis clinical isolates resistant to H versus drug susceptible isolates.

Methods Individual MICs of the strains were studied. Both combinations were studied crossing 7 concentrations of each antibiotic (including their MIC). The inoculum was of 104CFU/ml. All cultures were performed with the proportional method in Middlebrook 7H11 medium and incubated 4 weeks. On analysing the results of the combinations, the fractional inhibitory concentration (FIC) was interpreted: FIC≤0.5, synergistic activity; FIC from 1-4 indifference and FIC>4 antagonistic activity.

Results H+R+E Combination: 11 H resistant isolates were studied: 4 (36%) isolates had MIC=52µg/ml and 7 (64%) had MIC=0.8µg/ ml. 9 drug-susceptible isolates were also studied. Among the 20 isolates the individual MIC for R ranged from 1-2 µg/ml, from 2.5-5 µg/ml for E, being 0.05µg/ml for H in susceptible isolates. In H-resistant isolates, the MIC in combination for H and R decreased up to 3 dilutions (average) respect to their individual MIC. A lesser decrease (2-3 dilutions) was observed for E. In H-resistant isolates, 9/11 (81.1%) showed synergistic activity while 2/11 (18.18%) of the resistant isolates indicated indifference (FIC index =0.7). MICs in combination of susceptible strains decreased an average of 2 dilutions compared to their individual MIC. The susceptible strains had FIC indices ≥ 0.748. O+R Combination: 21 isolates were studied:11 had MIC≤1µg/ml, 4 MIC=1-6.4 µg/ml and 6 MIC>6.4. 9 drug-susceptible isolates were studied. Individual MICs for R ranged from 1-2, with 1 for O. MICs in combination of all strains were the same or decreased up to 1 dilution with regard to their individual MIC. The combination of OR in 21 strains did not show synergism or antagonism in either the resistant or the susceptible strains being the FIC values mainly 1.5.

Conclusions 1)These results suggest that in strains resistant to H with an MIC of 0.8µg/ml in vitro, the standard antibiotic regimen, including H, would be effective due to the possible compensatory effects of R and E. 2) Although strains resistant to H with an MIC of 51.2 µg/ml showed synergism, these strains would be within the range of resistance. 3)The combination of HRE against susceptible strains and that of OR did not show synergism probably because of high individual bactericide action.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 179 PP-108

TOBRAMYCIN-CLARITHROMYCIN COMBINATION ON Mycobacterium tuberculosis CLINICAL ISOLATES

Karolien Stoffels1, Hamidou Traore2, Raymond Van Hoof3 and Maryse Fauville-Dufaux1 1 - National Reference Laboratory of Tuberculosis and Mycobacteria, Scientific Institute of Public Health, Department Institut Pasteur, Rue Engeland 642, 1180 Brussels, Belgium 2 - Laboratoires SMB, Rue de La Pastorale 26-28, 1080 Brussels, Belgium 3 - National Reference Laboratory of Resistance to Aminoglycosides, Scientific Institute of Public Health, Department Institut Pasteur, Rue Engeland 642, 1180 Brussels, Belgium

Objectives This study investigated the in vitro susceptibility of 25 Mycobacterium tuberculosis clinical isolates to two well-known drugs, tobramycin (TM) and clarithromycin (CL). The effect of both drugs administered together was also investigated to detect a possible synergistic effect.

Methods MIC of isolates with variable resistance profiles was determined by the radiometric BACTEC 460 TB and was defined as the minimal antibiotic concentration for which at least 99% of the mycobacterial population growth was inhibited. The influence of TM on the MIC of CL was interpreted by Fractional Inhibitory Concentration (FIC).

Results The median MIC for both TM and CL was 8 µg/ml (range: 2 to 8 µg/ml and ≤2 to >16 µg/ml respectively). For 36% (9/25) of the tested isolates a decrease of the MIC of CL by a single or twofold dilution was observed (FIC ≤ 0,5) when a sub- inhibitory concentration of TM was added. No antagonistic effect (FIC > 4) was observed. Similar results were observed for isolates susceptible or resistant to first-line antituberculosis drugs.

Conclusion Although the MICs for CL and TM seem high compared to conventional anti-tuberculosis drugs, these antibiotics should not immediately be ruled out as clinically insignificant. Indeed, after administration of 300mg aerosolised TM, antibiotic concentrations in the epithelial lining fluid are higher than 10 times the median MIC observed for the TB isolates tested in this study (8µg/ml). Oral administration of 500 mg CL twice daily leads to a peak concentration of 13,50 +/- 3,3 µg/g in the lungs, 505 ± 293 µg/ml in alveolar cells and 34 ± 5 µg/ml in epithelial lining fluid, thus all exceeding the MIC of 8 µg/ml. Promising new drug delivery systems such as the Dry Powder Inhaler allow achieving very high local antibiotic concentration, e.g. 7 times higher for TM compared to aerosol administration and thus far beyond the MIC of resistant isolates. These results suggest that both drugs should be investigated further as potential adjuncts to the difficult treatment of multi-drug resistant tuberculosis where other alternatives have failed.

180 ESM 2009 PP-109

PREVALENCE OF EFFLUX-MEDIATED RIFAMPICIN RESISTANCE IN Mycobacterium tuberculosis CLINICAL ISOLATES

Carrie K. W. Au-Yeang, T. K. Au, Edward W. C. Chan, Raphael C. Y. Chan Department of Microbiology, The Chinese University of Hong Kong, The Prince of Wales Hospital, Shatin, New Territories, Hong Kong

Rifampicin is one major ingredient in the cocktail-regimen used in treatment of Mycobacterium tuberculosis (MTB) infection. Although mutations in the rpoB gene are considered the basis of rifampicin resistance, we noted that a significant proportion of local resistant cases could not be attributed to mutations. Alternative mechanisms such as drug efflux have been proposed. In order to evaluate the role of drug efflux mechanisms in mediating rifampicin resistance in MTB, we examined the prevalence of efflux activities in rifampicin resistant isolates using three efflux inhibitors: reserpine, carbonyl cyanide chlorophenylhydrazone and verapamil. Forty-two rifampicin resistant and nine drug susceptible MTB clinical isolates were studied. The minimum inhibitory concentration (MIC) values for rifampicin were determined in the presence and absence of efflux inhibitors. The magnitude of MIC reduction for each efflux inhibitor and the prevalence of efflux-mediated resistance were examined. We found that among the three efflux inhibitors tested, significant MIC reduction, ≥ 2-fold MIC decrease, was observed only for verapamil. 61% (31/51) of the test isolates had an MIC reduction between 2 to 8-fold in the presence of verapamil. This verapamil-sensitive efflux-mediated MIC reduction effect was much more apparent in rifampicin resistant isolates than the drug susceptible controls: 71% (30/42) versus 11% (1/9). Likewise, this phenomenon was more prevalent in isolates resistant to 3 - 5 anti-tuberculosis drugs than isolates resistant to 1 - 2 drugs: 77% (24/31) versus 55% (6/11). This data support the notion that drug efflux systems contribute significantly to rifampicin resistance in MTB clinical isolates and highlight the need for determining the extent by which these systems contribute to resistance to other anti-tuberculosis drugs. This study is supported by a grant (08070292) from Research Fund for the Control of Infectious Diseases.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 181 PP-110

INTRA AND EXTRACELLULAR ACTIVITY OF RUTHENIUM COMPLEXES AGAINST Mycobacterium tuberculosis AND THEIR CYTOTOXICITY

Leite, Clarice Queico Fujimura 1, Pavan, Fernando Rogério 1, Maia, Pedro I da S 2, Deflon, Victor M 2, Sato, Daisy Nakamura 3, Azevedo, Alzir A 4, Poelhsitz, Gustavo V 4, Leite, Sérgio Roberto Andrade 1, Franzblau, Scott G 5 1 - São Paulo State University 2 - University of São Paulo 3 - Adolfo Lutz Institute 4 - Federal University of São Carlos 5 - University of Illinois

Introduction Worldwide, tuberculosis (TB) remains the most frequent and important infectious disease causing morbidity and death. One-third of the world’s population is infected with Mycobacterium tuberculosis (MTB), the etiologic agent of TB. The World Health Organization estimates that about eight million new TB cases occur annually. The pulmonary TB (most common form of TB), is highly contagious and has been increasing in incidence in many areas, not only in developing countries but also in industrialized countries. The global resurgence of TB and the rapid emergence of MDR-TB, underscore the importance of the development of new antituberculous drugs.

Objectives Objecting to find new compounds with high activity against TB, we determined the cytotoxicity, and intra/extracellular anti-M. tuberculosis activity of 29 new compounds involving different class of ligands such as diimines, phosphines and Schiff bases with ruthenium.

Material and Methods As analytical methods, three standardized techniques were used: 1- in vitro Microplate Rezarurin Assay (REMA) for detection of minimal concentration of compounds necessary to inhibit 90% of bacillary growth (PALOMINO et al., 2002), 2- Cytotoxicity (IC50) of compounds against mamarian cells (AHMED et al., 1994) and 3- Determination of compounds activity against M. tuberculosis internalized in a macrophage cells (SNEWIN et al., 1999).

Results and Conclusion From the 29 compounds analyzed, 7 of them containing the ruthenium, were qualified as promising anti-TB agents. These complexes presented inhibitory activity ranging of 0.25 – 3.9 μg/mL, whose values are better than some drugs commonly used in the TB treatment, low cytotoxicity (IS > 10) and intracellular inhibitory activity high than 70%.

182 ESM 2009 PP-111

ANTI-MYCOBACTERIUM TUBERCULOSIS ACTIVITY OF THIOSEMICARBAZONES, SEMICARBAZONES AND HYDRAZONES

Leite; Sergio 1, Pavan; Fernando 2, Maia; Pedro 3, Deflon; Victor 3, Batista; Alzir 4, Sato; Daisy 5, Franzblau; Scott 6, Leite; Clarice 2 1 - Universidade Estadual Paulista, Instituto de Química 2 - Universidade Estadual Paulista, Faculdade de Ciências Farmacêuticas 3 - Universidade de São Paulo, Instituto de Química de São Carlos 4 - Universidade Federal de São Carlos, Departamento de Química 5 - Instituto Adolfo Lutz, Unidade de Ribeirão Preto 6 - University of Illinois at Chicago, College of Pharmacy

The aim of this study was to identify a candidate drug for anti-tuberculosis therapy development from previously synthe- sized thiosemicarbazones, semicarbazones and hydrazones, comprising a total of 17 compounds. The Minimal Inhibitory Concentration (MIC) of these compounds, determined by the resazurin reduction method, was investigated in order to determine their in vitro antimycobacterial activity against Mycobacterium tuberculosis. In vitro cytotoxicity values (IC50) of the same compounds were determined on J774 cells to establish a selectivity index (SI = IC50/MIC). Lower values of MIC were found for four thiosemicarbazones and four hydrazones, namely: 2-acetylpyridine N4 (etil) thiosemicarbazone; 2-acetylpyridine N4 (cyclohexyl) thiosemicarbazone; di-2-pyridyl ketone N4 (phenyl) thiosemicarbazone; 2-acetylpyridine morpholyl thiosemicarbazone; mono benzoylacetone isonicotinoyl hydrazone; mono-acetylacetone isonicotinoyl hydrazone; di-2-pyridyl ketone isonicotinoyl hidrazone; di-2-pyridyl ketone thiophene hidrazone (MIC values ranging from 0.78 to 6.25 μg/mL). All the compounds presented very low cytotoxicity, with the exception of 2-acetylpyridine morpholyl thiosemicarbazone (IC50 ≤ 3.9 μg/mL and SI ≤ 5). The results obtained with the other 7 compounds (SI ranging from 100 to 800) qualify them as candidates for anti-TB drugs, once their in vitro results are comparable to some of “first line” and “second line” drugs commonly used in the TB treatment.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 183 PP-112

METHODS FOR ASSESSMENT OF ETHIDIUM BROMIDE TRANSPORT ACROSS Mycobacterium smegmatis CELL WALL

Jorge Ramos1*, Liliana Rodrigues1,2, Isabel Couto1,3, Leonard Amaral1,2 and Miguel Viveiros1 1 - Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Lisboa, Portugal 2 - UPMM, IHMT/UNL, Lisboa, Portugal 3 - Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e Tecnologia, UNL, Caparica, Portugal *Correspondig author: Jorge Ramos, Unit of Mycobacteriology, Instituto Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Rua da Junqueira 96, 1349-008 Lisboa, Portugal. Tel: +351 21 3652600; Fax: +351 21 3632105; E-mail: [email protected]

Active efflux systems and reduced cell wall permeability are considered to be the main causes of mycobacterial intrinsic resistance to many antimicrobials. Although several mycobacterial efflux pumps have already been described, their role in drug resistance is not yet fully understood. Recent studies showed that both LfrA and MspA, the main efflux pump and porin in M. smegmatis, respectively, are involved in reduced susceptibility to several antimicrobials. We have compared the M. smegmatis wild-type strain mc2155 with LfrA and MspA M. smegmatis deleted mutants, for their ability to extrude ethidium bromide (EtBr), a known efflux pump substrate, under different energy conditions and in the presence or absence of efflux pumps inhibitors (EPIs), by (i) a 96 well microplate screening assay with the mycobacterial cells grown in Middlebrook 7H9 with 10% of OADC in presence of increasing concentrations of EtBr and different concentrations of the EPIs and the plates examined with a UV transilluminator and photographed after 24 hours of incubation; and (ii) a semi-automated fluorimetric method that detects efflux on a real time basis during a period of 30 minutes. The EPIs employed were chlorpromazine, thioridazine, carbonyl cyanide m-chlorophenylhydrazone and verapamil. The efflux activity detected for each strain by these two methods was then correlated with resistance to several antibiotics (ATBs), by the determination of their minimal inhibitory concentrations in the presence or absence of the EPIs. The ATBs tested were streptomycin, isoniazid (INH), rifampicin (RIF), ethambutol (ETB), amikacin, ciprofloxacin (CIP) and clarithromycin (CLR). In the absence of the major porin of M. smegmatis, MspA, it was observed that accumulation of EtBr decreased and the cells became more resistant to several ATBs. On the other hand, the mutant for the major efflux pump LfrA showed increased accumulation of EtBr. This strain also presented increased susceptibility to EtBr, INH, RIF, ETB, CIP and CLR. These results show that MspA is an important channel for entrance of quaternary ammonium compounds and ATBs and that the pump LfrA is involved in low-level resistance to several ATBs and quaternary ammonium compounds in M. smegmatis.

184 ESM 2009 PP-113 the human macrophage as a model to select compounds active against MDR/XDR-TB

Marta Martins1,2,*, Miguel Viveiros1,3, Isabel Couto1,4 and Leonard Amaral1,2,3. 1 - Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Lisbon, Portugal 2 - UPMM, IHMT/UNL, Lisbon, Portugal 3 - COST ACTION BM0701 (ATENS) 4 - Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e Tecnologia, UNL, Caparica, Portugal * Corresponding author: Unit of Mycobacteriology and UPMM, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Rua da Junqueira, 96, 1349-008, Lisbon, Portugal; Telf: +351213652600; Fax: +351213632105; e-mail: [email protected]

The emergence of Multi- and Extensively-Drug Resistant Mycobacterium tuberculosis (MDR/XDR-TB) represents a major threat to public health worldwide. Both infections result in high mortality, especially if the patient is co-infected with HIV. The selection of therapy for these multi-drug resistant infections is limited, and for most situations, ineffective as many of these strains are untreatable with the available drugs. Thus, there is an urgent need to design and develop new compounds against drug resistant M. tuberculosis that are effective within the main target of this infection, the human macrophage. We have recently demonstrated that efflux pumps inhibitors are active against mycobacteria, by enhancing the killing activity of the human macrophage and may represent an alternative to the conventional antibiotherapy for the treatment of the MDR/XDR-TB infections. From previous studies we have demonstrated that thioridazine (TZ) enhances the killing of MDR-TB phagocytosed by human macrophages. However, the mechanism of action of TZ on these cells is not fully understood. We have studied the activity of TZ, several of its derivatives, organosilicon (SILA) compounds and other known inhibitors of K+ and Ca2+ transport (ouabain, reserpine and verapamil) on macrophages infected with MDR-TB and XDR-TB. After phagocytosis, the compounds were added to the macrophage cultures. Following incubation cells were lysed and the intracellular bacterial concentration determined. Our results demonstrate that TZ, three of its derivatives and one SILA compound (SILA 421) enhanced substantially the macrophage killing activity. The killing activity of neutrophils is correlated with the K+ availability, which is dependent upon transport processes affected by agents that inhibit Ca2+-activated K+ pumps. Based on this and on our results, we postulate that the enhance- ment of the macrophage killing activity by these compounds could be due to the inhibition of Ca2+ and K+ transport that promotes the activation of hydrolases and the killing of intracellular bacteria. A model describing the sequence of events that lead to the killing of intracellular bacteria will be presented. Moreover, ��the ex-vivo testing of compounds using patient’s own macrophages in the clinical TB laboratory might allow screening the most effective compounds against MDR/ XDR-TB providing the basis for the intelligent selection of drugs to be used in the therapy of these infections.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 185 PP-114

IN VITRO ACTIVITIES OF JPC 2067 ALONE AND IN COMBINATION WITH SMX AGAINST NOCARDIA SPECIES

Michael Cynamon, Swagatam Mookherjee, Carolyn Shoen Veterans Affairs Medical Center, Syracuse, New York, USA E-mail: [email protected] FAX: 315-425-4871

Background JPC 2056, a biguanide prodrug of JPC 2067, a dihydrotriazine DHFR inhibitor, is being developed as an antimalarial therapeutic. Previously we demonstrated that earlier compounds related to JPC 2067 had promising activities in vitro alone and in combination with sulfamethoxazole (SMX) against nocardia species. JPC 2067 is active against M. tuberculosis, M. kansasii, and M. marinum at ≤ 1µg/ml. The purpose of the present study was to evaluate the in vitro activities of JPC 2067 alone and in combination SMX against a group of clinical nocardia isolates.

Methods JPC 2067 was provided by Jacobus Pharmaceutical Co., Princeton, NJ. SMX was purchased from Sigma Chemical Co, St. Louis, MO. Each drug was dissolved in DMSO at a final concentration of 1 mg/ml. Aliquots were frozen at -200C. Drugs were thawed prior to testing and diluted in modified 7H10 broth (pH 6.6; 7H10 agar formulation with agar and malachite green omitted) with 10% OADC enrichment and 0.05% Tween 80. JPC 2067 and SMX were tested alone from 64µg/ml – 0.06µg/ml. When tested together SMX was evaluated at fixed concentrations of 1 µg/ml and JPC at 16µg/ml – 0.015µg/ ml. Twenty eight nocardia isolates (from the ATCC and clinical isolates provided by B. Body and B. Forbes) were used in the study. An in vitro broth dilution method similar to that defined by CLSI was utilized.

Results:

The MIC50 and MIC90 for JPC 2067, SMX and the combination (SMX fixed at 1µg/ml) were 0.125 µg/ml and 4 µg/ml, 16 µg/ml and 32 µg/ml, and 0.03 µg/ml and 2 µg/ml respectively.

Conclusions JPC 2067 was more active than the previously tested dihydrotriazine analogs against nocardia. The addition of SMX had a modest but consistent effect on lowering the JPC 2067 MIC. It is likely that this effect would be more pronounced if the concentration of SMX was increased to perhaps10 µg/ml (a readily achieved serum level for this agent). JPC 2067 alone and in combination should be evaluated in animal models of both nocardial and mycobacterial infection to understand the clinical potential of these agents.

186 ESM 2009 PP-115

Nosocomial TB in a laboratory setting

Jaime M S Nina1,2,3 1 - Instituto Nacional de Saúde Doutor Ricardo Jorge 2 - Universidade Nova de Lisboa 3 - Hospital Egas Moniz

Abstract TB is recognized as a major cause of morbidity and mortality worldwide. Its easily transmissibility is also generally recognized, both at the family level, in the household, at the place of work and inside health care facilities. This last way of transmission, properly called nosocomial transmission, has been suspected for long time, and was formally demonstrated in the ward, both among patients, and health care workers. Several professional bodies and other institutions, both at the national and international level, produced guidelines trying to minimize TB transmission to health care workers inside wards and emergency services. Furthermore several countries produced legislation to protect health care workers against nosocomial TB, and/or included TB in the list of professional diseases or hazards to health care workers. However, much less attention has been given to the TB transmission potential to laboratory workers. Even if several countries moved laboratory work with live TB samples to LSB-3 facilities, the evidence on which to base this decision is thin, and no systematic study has been published. Herein are presented three cases of nosocomial transmission inside laboratory settings, in Lisbon. These cases cover all spectra of professional differentiation, from basic level auxiliary personnel, to a laboratory technician, and to a micro- biologist physician. In one case the way of transmission was a very common kind of laboratory accident, in another a common inattention, and in the last one no specific way of transmission was found. One of the cases was found to be a MDX TB. In conclusion, the trend to carry out all routine work with live TB samples only inside a BSL-3 facility seems a right one, and so it is fully justified. Also justified would be the inclusion of health laboratory workers in the legislation that provide safety measures and insurance cover to health care workers.

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 187

Author Index

Abadia E Beltrán M Cubillos A Furlaneto IP Jahromi NS Abreu C Biet F Cvetnic Z Furtado C Jansone I Afonso A Boehme C Cynamon M Gagneux S Janssen HG Aguilar D Boeree M Dafae F Gaile I João I Ahmadi M Bonte L David S Gama JB Joloba M Ahmed A Borile C De Bock A Gao Q Joloba ML Ahmed I Borroni E De Cruz K Garcia MJ Jordão L Åkerström M Boschiroli ML De Gispert FX García-Cañas A Joshi S Akol J Bourdon E De Haas P Gavin P Julián E Akpaka PE Braga JE De la Hoz F Gegia M Julián EG Al Balushi L BragaR De Sousa MS George AG Jureen P Al Busaidi S Brankova N Deflon V Gharbia S Kaal E Al- Mahruqi S Brisse Smangenot S Deflon VM Giampaglia CS Kahlmeter G Albarral MIP Brosch R Dekhuijzen R Gicquel B Kam KM Alcaide F Brown T Del Portillo P Gil MJ Kanavaki S Aldashev A Brum L Del Val-Romero B Gilpin CM Kang HS Algamdi S Brzostek A Dementieva A Giske C Kantor I Al-Hajoj S Bwanga F Den Hertog A Gitti Z Kapata N Ali AB Cabral J Di Giulio B Goguet de la Salmonière YOL Karabela S Ali MS Cacho J Dias A Goldfeder LC Karacali A Ali Veleyati A Caldas C Diogo J Golec M Kargar M Allix-Béguec C Cambau E Diwan V Gomes H Karoui C Al-Maniri AA Cañete C Docx S Gomes HM Katalinic-Jankovic V Almeida da Silva P Cano I Drobniewski F Gomes MH Katalinic-Jankovic V Almeida EA Cardoso N DuarteR Gonçalves H Kayar B Al-Omari R Cardoso RF Duvnjak S González Torralba A Kayar Mb Alonso C Cardoso S Dziadek B González-Martín J Kazempour M Al-Rawas O Carmona JA Dziadek J Grce M Kern WV Alves A Carvalho C Echemendía M Greib C Khan AJ Amado J, Carvalho F Ehricht R Guillard B Khechinashvili G Amaral L Carvalho MA Eisenach K Gutiérrez C Kim JH Amondi Ouma N Carvalho T Elmoula IF Gutierrez J Klatser P Amorim A Casal M Etwom A Gutierrez MC Koeleman M Andersson E Cassone A Eum SY Gutierrez-Aroca JB Köksal F Ängeby K Castro AG Fajfar N Hadadi M Kolk A Anoosheh S Causse M Falkinham, III, JO Haenn S Konstantinidou E Anthony R Chan Chiu Y Farnia P Haile M Kontos F Arnold C Chan CYR Fattorini L Haroun RZ Kopher K Au TK Chan EWC Fauville-Dufaux M Havelkova M Kosmadakis G Au-Yeang CKW Chan RCY Felix C Hepple P Kritski A Awad C Chan WCE Ferme D Herbawi M Krt B Ayles H Chau CH Fernandes P Hernández J Kuan HO Azevedo AA Cheruiyot C Fernandes SJ Hernández-Pando R Kubin M Baboolal S Cho E-J Ferreira A Hillemann D Kuijper S Babu Okatch F Cho SN Ferreira C Hirata MH Labarre M Bachiiska E Chryssanthou E Ferreira D Hirata RDC Lai WMR Balacó I Cirillo D Ferreira S Hoffner S Langerak E Balbin JA Cirillo DM Ferro RS Homolka S Lazaro E Baldan R Clivillé R Feuerriegel S Honisch C Leão SC Balmoi F Cochard T Fey F Honscha G Lee H Barbe V Codina G Figueira R Hoosen A Lee J Baritaki M Coelho R Filippini P HubansC Lee J-I Baritaki S Coll P Fissette K Hwang SH Leimane V Barrera L Conceição EC Fiuza de Melo F Ibrahim TA Leite C Barry III CE Correa N Frade R Ichijo T Leite CQF Bartu V Correia-Neves M Fraga AG Imperiale B Leite S Batista A Costa ARF Frangopoulos F Ingham C Leite SRA Baumanis V Couto I Franz S Ioannidis P Leite SRA Bauskenieks M Couto S Franzblau S Isakova J Lemos S Bazigos S Crews V Franzblau SG Ivanov A Lemus D Beaty PS Cruz A Fung SL Izumi Y Levina K European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 189 Levterova V Monecke S Pellegrin JL Sancho L Van der Stuyft P Lezcano MA Monge I Perdigão J Sandoval A van der Wel N Lima EJC Moniz-Pereira J Pereira DR Santos ACB Van Hoof R Lima KVB Monteiro G Pereira E Santos C van Ingen J Locht C Montemayor M Pereira Miguel J Santos R Van Soolingen D Lockwood D Montoro E Pérez Meixeira A Saraiva M Varaine F Logarinho E Mookherjee S Perkins M Sardinha E Varghese B Lopes ML Morcillo N Pfeltz R Sardinha T Vasconcelos O López AG Morgan K Pfyffer G Sarmento A Velayati A López B Mosko M Pimentel M Sato D Velayati AA Lopez-Calleja AI Mota M Pinheiro MD Sato DN Via LE Loureiro C Moulin L Poelhsitz GV Schön T Viallard JF Lucas F Moure R Pole I Secanella SP Viana BHJ Luo T Moyoyeta M Portaels F Seif S Viana-Niero C Luquin M Muchwa C Portugal C Shamputa IC Villar M Lyashchenko K Mugerwa R Portugal I Sharaf-Eldin GS Villela G Lyberopoulos P Mugyenyi P Possuelo L Shikama M-L Viveiros M Macedo R Müllerova M Post E Shinnick T Vladimirov K Machado D Mumbowa F Prata P Shinnick TM Von Groll A Madruga M Munga Waweru P Proença F Shoen C Vultos TD Maia P Murcia M Qazi F Siddiqi S Wang S Maia PIS Musunsa A Quieng MD Silva A Warns M Maio JN Muvwimi M Racic I Silva C Watson C Mak KX Mwamba P Radomski N Silva F Weizenegger M Malagari AI Mwanza W Ramos J Silva K Werngren J Malaquias A Nagiyev T Ramos Martos Silva M Westman L Malaspina AC Nascimento I Ramos MH Silva P Willery E Mallard K Nasu M Ramoutar D Silva S Winkler S Marceau M Navarro A Raposo A Simões MF Wong AP Markova N Neo ZY Rasolofo V Sing LH Yamaguchi N Marques M Neonakis IK Rastogi N Singh JPN Yamane N Martín A Niemann S Rauzier J Skenders G Yan SW Martin C Nikolaou S Refrégier G Slickers P Yates M Martín-Casabona N Nina J Régis M Sola C Yew WW Martinez-Martinez L Nogueira C Reniero A Somoskövy A Yip CW Martins M Nogutia EN Rey E Sousa AS Yubero J Martins MC Noroozi J Reyes A Sousa C Yula E Martins TG Noruzi J Ribeiro JN Sousa G Yzquierdo S Masjedi M Novoa-Cain J Richter E Sousa JG Zaitseva E Masjedi MR Nowroozi J Ridell M Sovhozova N Zaldumbide MA Matic I Nuak J Riley LW Soyal A Zambrano MM Matos G Nyirenda CN Ritacco V Spandidos DA Maugein J Oberhauser B Ritmeijer K Spicic S Zdelar-Tuk M Mazarrasa CF Obrovac M Robledo J Stoffels K Zellweger J-P Mbulo G Obrovac M Rocha G Sturegård E Zenhorst R McNerney R Ocepek M Rodrigues A Suffys PN Zerolo FJ Mdivani N Oelemann MC Rodrigues F Supply P Zerva L Médigue C Ojeda P Rodrigues L Svensson E Zhang J Mei J Oliveira P Rodrigues S Tabarsei P Zmak L Mello FAF Omar AR Rodríguez-Güell E Tajeddin E Zolnir - Dovc M Mendes AC Oral Zeytinli U Rohde KH Takiff H Žolnir Dov- M Mendes NH Orikiriza P Rosales J Tancredo L Mendes NH Orozco H Roura-Mir C Tang YW Zozio T Menendez MC O’Sullivan D Ruimy R Tap J Mestre O Paasch F Ruiz P Tavares M Meyers WM Palaci M Rumijowska-Galewicz A Tavares Magalhães A Mézard M Palomino JC Rüsch-Gerdes S Teles JMM Michel G Panaiotov S Russell DG Telles MAS Mihailelis E Pando RH Saaed NS Thibault V Milho C Pandolfi JRC Sabino A Tonjum T Milián Y Papaventsis D Şahan Kipalev A Torrado E Millan I Papiris S Said H Tortoli E Millet J Pardini M Sainti A Traore H Min JH Park SK Salas S Tudó G Miotto P Pate M Saluotsa M Turner C Mirabal N Paulo C Salvadó M Ueki SYM Miranda A Pavan F Salvignol G Valcheva V Miyagi-Shiohira C Pavan FR Sampaio D Valdés I Miyata M Pawelczyk J Samper S Valente A Moilleron R Pedrosa J Samutela M Valente F Mokrousov I Peeling R Sánchez-Concheiro M Valente I

190 ESM 2009 Levterova V Monecke S Pellegrin JL Sancho L Van der Stuyft P Lezcano MA Monge I Perdigão J Sandoval A van der Wel N Lima EJC Moniz-Pereira J Pereira DR Santos ACB Van Hoof R Lima KVB Monteiro G Pereira E Santos C van Ingen J Locht C Montemayor M Pereira Miguel J Santos R Van Soolingen D Lockwood D Montoro E Pérez Meixeira A Saraiva M Varaine F Logarinho E Mookherjee S Perkins M Sardinha E Varghese B Lopes ML Morcillo N Pfeltz R Sardinha T Vasconcelos O López AG Morgan K Pfyffer G Sarmento A Velayati A López B Mosko M Pimentel M Sato D Velayati AA Lopez-Calleja AI Mota M Pinheiro MD Sato DN Via LE Loureiro C Moulin L Poelhsitz GV Schön T Viallard JF Lucas F Moure R Pole I Secanella SP Viana BHJ Luo T Moyoyeta M Portaels F Seif S Viana-Niero C Luquin M Muchwa C Portugal C Shamputa IC Villar M Lyashchenko K Mugerwa R Portugal I Sharaf-Eldin GS Villela G Lyberopoulos P Mugyenyi P Possuelo L Shikama M-L Viveiros M Macedo R Müllerova M Post E Shinnick T Vladimirov K Machado D Mumbowa F Prata P Shinnick TM Von Groll A Madruga M Munga Waweru P Proença F Shoen C Vultos TD Maia P Murcia M Qazi F Siddiqi S Wang S Maia PIS Musunsa A Quieng MD Silva A Warns M Maio JN Muvwimi M Racic I Silva C Watson C Mak KX Mwamba P Radomski N Silva F Weizenegger M Malagari AI Mwanza W Ramos J Silva K Werngren J Malaquias A Nagiyev T Ramos Martos Silva M Westman L Malaspina AC Nascimento I Ramos MH Silva P Willery E Mallard K Nasu M Ramoutar D Silva S Winkler S Marceau M Navarro A Raposo A Simões MF Wong AP Markova N Neo ZY Rasolofo V Sing LH Yamaguchi N Marques M Neonakis IK Rastogi N Singh JPN Yamane N Martín A Niemann S Rauzier J Skenders G Yan SW Martin C Nikolaou S Refrégier G Slickers P Yates M Martín-Casabona N Nina J Régis M Sola C Yew WW Martinez-Martinez L Nogueira C Reniero A Somoskövy A Yip CW Martins M Nogutia EN Rey E Sousa AS Yubero J Martins MC Noroozi J Reyes A Sousa C Yula E Martins TG Noruzi J Ribeiro JN Sousa G Yzquierdo S Masjedi M Novoa-Cain J Richter E Sousa JG Zaitseva E Masjedi MR Nowroozi J Ridell M Sovhozova N Zaldumbide MA Matic I Nuak J Riley LW Soyal A Zambrano MM Matos G Nyirenda CN Ritacco V Spandidos DA Maugein J Oberhauser B Ritmeijer K Spicic S Zdelar-Tuk M Mazarrasa CF Obrovac M Robledo J Stoffels K Zellweger J-P Mbulo G Obrovac M Rocha G Sturegård E Zenhorst R McNerney R Ocepek M Rodrigues A Suffys PN Zerolo FJ Congress organisers Mdivani N Oelemann MC Rodrigues F Supply P Zerva L Médigue C Ojeda P Rodrigues L Svensson E Zhang J Mei J Oliveira P Rodrigues S Tabarsei P Zmak L Mello FAF Omar AR Rodríguez-Güell E Tajeddin E Zolnir - Dovc M Mendes AC Oral Zeytinli U Rohde KH Takiff H Žolnir Dov- M Mendes NH Orikiriza P Rosales J Tancredo L Mendes NH Orozco H Roura-Mir C Tang YW Zozio T Menendez MC O’Sullivan D Ruimy R Tap J Mestre O Paasch F Ruiz P Tavares M Meyers WM Palaci M Rumijowska-Galewicz A Tavares Magalhães A Mézard M Palomino JC Rüsch-Gerdes S Teles JMM Skyros-Congressos Michel G Panaiotov S Russell DG Telles MAS Av.Antunes Guimarães, nº 554 Mihailelis E Pando RH Saaed NS Thibault V Milho C Pandolfi JRC Sabino A Tonjum T 4100-074 Porto Milián Y Papaventsis D Şahan Kipalev A Torrado E Millan I Papiris S Said H Tortoli E Tel.: (+351) 226 165 450 Millet J Pardini M Sainti A Traore H Min JH Park SK Salas S Tudó G Fax: (+351) 226 189 539 Miotto P Pate M Saluotsa M Turner C E-mail: [email protected] Mirabal N Paulo C Salvadó M Ueki SYM Miranda A Pavan F Salvignol G Valcheva V Miyagi-Shiohira C Pavan FR Sampaio D Valdés I Miyata M Pawelczyk J Samper S Valente A Moilleron R Pedrosa J Samutela M Valente F Mokrousov I Peeling R Sánchez-Concheiro M Valente I

European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal 191