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Address:
Ospedale San Raffaele - Milano,
via Olgettina 60,
20132 Milano, Italia
Contacts:
Daniela Cirillo, MD, PhD (cirillo.daniela@hsr.it)
Elisa Tagliani, PhD (tagliani.elisa@hsr.it)
Andrea Cabibbe, PhD (cabibbe.andreamaurizio@hsr.it)
Supranational TB Reference Laboratory in Milan, Italy
Description
The Emerging Bacterial Pathogens Unit (EBPU) headed by Dr. Daniela Maria Cirillo was established in 2003 with the aim to perform advanced mycobacterial diagnosis, to support development of new tools for TB detection and to study the pathogenic mechanisms of DR-TB. The Unit is the Italian reference centre for molecular surveillance of MDR-TB, typing and testing drug susceptibility to new drugs Delamanid and Bedaquiline. The laboratory has been accredited by the local authority and ISO 9001:2008 certified. EBPU has been recognized as WHO SupraNational Reference Laboratory for TB, as WHO Collaborating Centre ITA98 and as ESCMID collaborative centre.
Structure and organization
EBPU has large Cat2-3 laboratories, fully equipped for cell culture and molecular biology including the Illumina MiniSeq. In addition, the staff has access to all core facilities present in campus: the genome with all NGS platforms and bioinformatics support, FACS, microscopy and animal facilities. The EBPU staff includes medical doctors, PhD, MS, specialists in mycobacteriology, MPH, technicians and bioinformatics. All staff is trained in providing support to high burden TB Countries and in different aspects related to TB diagnosis and control. All staff is trained on quality management. Language spoken are English, Italian, French, Portuguese and Spanish.
Services provided by the SRL
EBPU provides trainings, quality assurance monitoring and technical support on TB and TB/HIV collaborating activities. It supports countries in the preparation of DRS and Prevalence Survey protocol and budget jointly with WHO. The Unit is a reference centre for second level TB diagnosis using conventional and molecular tools including whole genome sequencing for drug resistance determination and phylogenetic/epidemiologic analysis. In addition, through FIND we serve as a liaison between basic and industrial research and maintains long standing collaborations with diagnostic manufacturer (STMicroelectronics, Hain GmbH, Cepheid, QIagen) in the development and validation of new diagnostic platforms for M. tuberculosis.
Microscopy
Light and fluorescence microscopy;
Capacity to prepare panels for microscopy EQA;
Regularly undergo to EQA for microscopy (by panel testing) (ECDC network).
Culture
MTB culture on solid (LJ, 7H10 and 7H11 agar) and liquid media (Middlebrook 7H9) by manual and automated methods ( BACTEC MGIT 960);
Regularly undergo to EQA for culture (by panel testing) (ECDC network).
Molecular tools
LPA for first and second line drugs (MTBDRplus and MTBDRsl assays);
Xpert MTB/RIF and XpertULTRA assays;
qPCR;
In house PCR for MTB/NTM identification and drug resistance detection;
MIRU-VNTR-24 and spoligotyping;
Regularly undergo to EQA for molecular testing (ECDC network);
Preparation of panel for EQA purposes (LPA and MIRU-VNTR-24).
DST
Phenotypic DST for first, second line drugs and new drugs (BDQ, Delamanid) on solid and liquid media;
MIC assay on plate or multi-well plates (e.g. ThermoFisher Sensititre™ plate, REMA/MABA);
Capacity to develop DST assay for new drugs;
Regularly undergo to EQA for DST (ECDC network and SRL network);
Preparation of panels for EQA purposes (for supported countries)
Sequencing
Target sequencing onclinical specimens and isolates (Sanger and NGS);
Whole Genome Sequencing (WGS) on Illumina NGS platforms (HiSeq, MiSeq and MiniSeq).
Regularly undergo to EQA for WGS (ECDC network).
Other
NTM identification and drug susceptibility testing;
IGRA test (QFT-GIT and QFT-Plus);
Macrophage infection by MTB and cell response evaluation.
Current links and partnerships outside of SRLN
EBPU collaborates with WHO-CC for TB/HIV in Brescia in the conduction of operational research on TB/HIV co-infection and with WHO-CC for TB and Lung Diseases in Tradate in the conduction of trainings for TB consultants. The Unit is part of European Reference Laboratory Network for TB (ERLN-TB) launched by the ECDC with the aim to support methods harmonisation within the EU/EEA, to develop EQA schemes and to provide training activities to ensure EU-wide capacity building. In addition, the Unit is part of global consortia aimed at understanding the genetic basis of drug resistance in MTB (i.e. ReSeqTB and CRyPTIC Project).
Additional information:
Research activities
The staff at SRL Milan is made up of over 19 people, including medical doctors, biologists specialized in clinical microbiology, laboratory technicians, PhDs in molecular biology and bioinformatics. All members are fully trained in laboratory quality assurance procedures recommended by WHO. One third of total personnel at the EBPU-SRL Milan has years of experience in working in and with high burden TB Countries, and in deep knowledge of the WHO End TB Strategy and WHO TB related policies and guidelines.
We conducted more than 50 independent missions and spent more than 700 days across over 20 countries (Albania, Argentina, Bulgaria, Burkina Faso, Chile, Djibouti, Eritrea, Eswatini, Ethiopia, Gabon, Ghana, India, Indonesia, Ivory Coast, Kosovo, Mauritania, Mexico, Mozambique, Nigeria, Pakistan, Senegal, Sierra Leone, South Africa, Tanzania, Tunisia, Turkey).
We support National Tuberculosis Programmes (NTPs) in the development of national TB programs, laboratory strategic plans and quality management and monitoring systems, in the revision and update of testing algorithms, in the implementation and roll-out of new TB diagnostics, and in the uptake and use of different tools for TB laboratory strengthening through technical assistance visits, remote support and training courses.
The SRL Milan is actively involved in supporting the drug resistance surveillance projects of multiple Countries (i.e. Ukraine, Bangladesh, Pakistan, Djibouti, Ivory Coast, Burkina Faso, Kosovo, Eritrea, Ethiopia, Mozambique, Eswatini) using either target sequencing or whole genome sequencing approach for the identification of mutations leading to anti-TB drug resistance. The Illumina MiniSeq platform is currently available in the laboratory in addition to the Illumina NextSeq500 and MiSeq available at the genome campus facility. The Unit has contributed to the development of web tools, bioinformatics pipelines and targeted sequencing protocols for delineating anti-TB drug resistance and genotyping from sequencing data. The Unit is assisting several NTPs and National Reference Laboratories (NRLs) in implementing sequencing capacity.
In collaboration with international non-profit organizations, diagnostics’ manufacturers and TB reference laboratories, we carry out research activities aimed at the development and performance evaluation of new molecular-based diagnostic platforms for TB, and DR-TB detection. We have contributed to the development and validation of microarray-based tools, line probe assays for first and second line anti-TB drugs including pyrazinamide. We have participated in multicenter accuracy studies of several molecular WHO-recommended rapid diagnostics (mWRDs) (e.g., Xpert MTB/RIF ULTRA, Xpert MTB/XDR, Truenat MTB), as well as next generation sequencing (NGS)-based assays for DR-TB prediction directly from sputum specimens (i.e., Deeplex Myc-TB).
In collaboration with other European Mycobacteriology Laboratories, we develop standardized procedures for phenotypic susceptibility testing of anti-TB drugs, including new and repurposed agents. We provide on-site and remote technical assistance to laboratory staff wanting to implement antimicrobial susceptibility testing to new and repurposed anti-TB agents, using WHO recommended critical concentrations.
We have contributed to the data analysis, the development and preparation of the catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance published by the WHO. The document is based on systematic analysis of a large collection of isolates of Mycobacterium tuberculosis complex with whole-genome sequencing and phenotypic drug-susceptibility testing. This data will support diagnostic developers, researchers and policy makers in the development and implementation of rapid drug susceptibility test assays needed to improve the management of TB patients at Country level, facilitate the rational use of new TB drug regimens, support drug regimen roll-out and better understand the global burden of drug-resistant TB.
Publications
Vargas R Jr, Freschi L, Spitaleri A, Tahseen S, Barilar I, Niemann S, Miotto P, Cirillo DM, Köser CU, Farhat MR. The role of epistasis in amikacin, kanamycin, bedaquiline, and clofazimine resistance in Mycobacterium tuberculosis complex. Antimicrob Agents Chemother. 2021 Aug 30:AAC0116421. ISSN: 0066-4804, doi: 10.1128/AAC.01164-21. Online ahead of print.
Alagna R, Cabibbe AM, Miotto P, Saluzzo F, Köser CU, Niemann S, Gagneux S, Rodrigues C, Rancoita PVM, Cirillo DM. Is the new WHO definition of extensively drug-resistant tuberculosis easy to apply in practice? Eur Respir J. 2021 Jul 1;58(1):2100959. ISSN: 0903-1936, doi: 10.1183/13993003.00959-2021. Print 2021 Jul.
Villa S, Tagliani E, Borroni E, Castellotti PF, Ferrarese M, Ghodousi A, Lamberti A, Senatore S, Faccini M, Cirillo DM, Codecasa LR. Outbreak of pre- and extensively drug-resistant tuberculosis in northern italy: urgency of cross-border, multidimensional, surveillance systems. Eur Respir J. 2021 May 28:2100839. ISSN: 0903-1936, doi: 10.1183/13993003.00839-2021
Hamada Y, Cirillo DM, Matteelli A, Penn-Nicholson A, Rangaka MX, Ruhwald M. Tests for tuberculosis infection: landscape analysis. Eur Respir J. 2021 Apr 19:2100167. ISSN: 0903-1936, doi: 10.1183/13993003.00167-2021.
Alagna R, Combary A, Tagliani E, Sawadogo LT, Saouadogo T, Diandé S, Ouedraogo F, Cirillo DM. Is deployement of diagnostic test alone enough? Comprehensive package of interventions to strengthen TB laboratory network: three years of experience in Burkina Faso. BMC Infect Dis. 2021 Apr. 13. ISSN: 1471-2334, doi.org/10.1186/s12879-021-06012-y.
Goscé L, Girardi E, Allel K, Cirillo DM, Barcellini L, Stancanelli G, Matteelli A, Hagphrast-Bidgoli H, Abubakar I. Tackling TB in migrants arriving at Europe's southern border. Int J Infect Dis. 2021 Mar 10:S1201-9712(21)00189-2. ISSN: 1201-9712, doi: 10.1016/j.ijid.2021.02.103.
Farooq HZ, Cirillo DM, Hillemann D, Wyllie D, van der Werf MJ, Ködmön C, Nikolayevskyy V. Limited Capability for Testing Mycobacterium tuberculosis for Susceptibility to New Drugs. Emerg Infect Dis. 2021 Mar;27(3):985-987. ISSN: 1080-6040, doi: 10.3201/eid2703.204418.
Mesfin AB, Araia ZZ, Beyene HN, Mebrahtu AH, Suud NN, Berhane YM, Hailu DT, Kassahun AZ, Auguet OT, Dean AS, Cabibbe AM, Cirillo DM. First molecular-based anti-TB drug resistance survey in Eritrea. Int J Tuberc Lung Dis. 2021 Jan 1;25(1):43-51. ISSN: 1027-3719, doi: 10.5588/ijtld.20.0558.
Cao Y, Parmar H, Gaur RL, Lieu D, Raghunath S, Via N, Battaglia S, Cirillo DM, Denkinger C, Georghiou S, Kwiatkowski R, Persing D, Alland D, Chakravorty S. Xpert MTB/XDR: A ten-color reflex assay suitable for point of care settings to detect isoniazid-fluoroquinolone-, and second-line injectable drug-resistance directly from Mycobacterium tuberculosis positive sputum. J Clin Microbiol. 2020 Dec 9: JCM.02314-20. ISSN: 0095-1137, doi: 10.1128/JCM.02314-20.
Schön T, Köser CU, Werngren J, Viveiros M, Georghiou S, Kahlmeter G, Giske C, Maurer F, Lina G, Turnidge J, van Ingen J, Jankovic M, Goletti D, Cirillo DM, Santin M, Cambau E; ESGMYC. What is the role of the EUCAST reference method for MIC testing of the Mycobacterium tuberculosis complex? Clin Microbiol Infect. 2020 Nov;26(11):1453-1455. ISSN: 1198-743X, doi: 10.1016/j.cmi.2020.07.037.
Alagna R, Sawadogo TL, Tagliani E, Combary A, Cirillo DM. Applying a Standardized Approach to Strengthen Performances of GeneXpert Networks programme: lessons learned from Burkina Faso, 2019. ERJ Open Res. 2020 Nov 16;6(4):00283-2020. ISSN: 23120541, doi: 10.1183/23120541.00283-2020.
Jouet A, Gaudin C, Badalato N, Allix-Béguec C, Duthoy S, Ferré A, Diels M, Laurent Y, Contreras S, Feuerriegel S, Niemann S, André E, Kaswa MK, Tagliani E, Cabibbe A, Mathys V, Cirillo D, de Jong BC, Rigouts L, Supply P. Deep amplicon sequencing for culture-free prediction of susceptibility or resistance to 13 anti-tuberculous drugs. Eur Respir J. 2020 Sep 17:2002338. ISSN: 0903-1936, doi: 10.1183/13993003.02338-2020.
Köser CU, Cirillo DM, Miotto P. How to optimally combine genotypic and phenotypic drug-susceptibility testing methods for pyrazinamide. Antimicrob Agents Chemother. 2020 Aug 20;64(9):e01003-20.ISSN: 0066-4804, doi: 10.1128/AAC.01003-20.
Tagliani E, Anthony R, Kohl TA, de Neeling A, Nikolayevskyy V, Ködmön C, Maurer FP, Niemann S, van Soolingen D, van der Werf MJ, Cirillo DM; ECDC molecular surveillance project participants. Use of a Whole Genome Sequencing-based approach for Mycobacterium tuberculosis surveillance in Europe in 2017-2019: an ECDC pilot study. Eur Respir J. 2020 Jul 30;2002272. ISSN: 0903-1936, doi: 10.1183/13993003.02272-2020.
Cabibbe AM, Spitaleri A, Battaglia S, Colman RE, Suresh A, Uplekar S, Rodwell TC, Cirillo DM. Application of targeted Next Generation Sequencing assay on a portable sequencing platform for culture-free detection of drug resistant tuberculosis from clinical samples. J Clin Microbiol. 2020 Jul 29:JCM.00632-20. ISSN: 0095-1137, doi: 10.1128/JCM.00632-20.
Ismail NA, Aono A, Borroni E, Cirillo DM, Desmaretz C, Hasan R, Mitarai S, Shakoor S, Torrea G, Kaniga K, Omar SV. A Multimethod, Multicountry Evaluation of Breakpoints for Bedaquiline Resistance Determination. Antimicrob Agents Chemother. 2020 Jul 13:AAC.00479-20. ISSN: 0066-4804, doi: 10.1128/AAC.00479-20.
Quaresima V, Naldini MM, Cirillo DM. The prospects for the SARS-CoV-2 pandemic in Africa. EMBO Mol Med. 2020 Apr 16. ISSN: 1757-4684 doi: 10.15252/emmm.202012488.
Trainings
The Unit provides practical trainings on the different molecular based technologies for MTB identification and drug-resistance determination (i.e. Xpert MTB/RIF Ultra assay, LPA for first and second line drugs) as well as sequencing and next generation sequencing using the Illumina / Oxford Nanopore Technologies platforms. We also provide trainings on conventional phenotypic based drug susceptibility testing using solid and liquid media, as well as microtiter plates (i.e. Sensititre™ Mycobacterium tuberculosis microtiter plate by ThermoFisher). Training are based on GLI/WHO/FIND resources adapted to the local need and are provided in English or French according to the preferences of the trainees.
We yearly provide a 5-days training with theoretical and practical sessions on molecular diagnosis of Drug Resistant- TB The training focuses on all techniques currently endorsed by WHO for the rapid diagnosis of tuberculosis and detection of antibiotic resistance as well as advanced molecular innovations (Whole Genome Sequencing) and is aimed at developing knowledge and skills needed for a successful implementation and optimal utilization of new TB diagnostic tools.
We collaborate with assisted Countries to develop tailored training courses aimed to strengthening laboratory and quality management systems.
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