Handbook On TB Laboratory Diagnostic Methods In The European Union

TECHNICAL DOCUMENTHandbook on TB laboratory diagnostic methods for the EUBackground and introductionFrancis DrobniewskiTuberculosis (TB) is a major cause of morbidity and mortality in Europe. High-quality laboratory diagnosis of TB isthe basis for both individual patient treatment and surveillance.In 2007, a survey of existing mycobacterial laboratory services and quality control practices throughout the EUconfirmed the key role of national reference laboratories (NRL) for TB and their services. The main conclusion ofthe survey was that a network of reference laboratories for tuberculosis could contribute to improving theperformance of mycobacterial laboratories in Europe.Based on these results, the European Reference Laboratory Network for Tuberculosis (ERLN-TB) was launched inJanuary 2010, with the aim of strengthening TB diagnostics in the European Union. ERLN-TB was funded andcoordinated by ECDC. One or two officially nominated reference laboratories from each Member State joined thenetwork, along with those in EEA countries and EU candidate countries.Following the success of the ERLN-TB network, in 2014 ECDC commissioned a renewal of the network (now calledEuropean Reference Laboratory TB-Network [ERLTB-Net]) involving the same centres but anticipating theparticipation of institutions from the EU enlargement countries in the longer-term.The three main goals of the new network are: to support the harmonisation of laboratory methods within theEU/EEA; develop External Quality Assurance (EQA) schemes and provide training activities within the network toensure EU-wide capacity-building for TB diagnostics. One of the main activities conducted by the network was thedevelopment of a handbook of key diagnostic methods for tuberculosis which was first published in 2011. Thisdocument represents the latest version of the handbook.The aim of this handbook is to provide network members and other laboratories involved in the diagnosis oftuberculosis, with an agreed list of key diagnostic methods and their protocols in various areas of TB diagnosis,ranging from microbiological diagnosis of active TB to the diagnosis of latent TB infection (LTBI). This handbookoffers a single source of reference by compiling all methods, with a strong focus on standard (reference) andevidence-based methods.The handbook will also contribute to the improvement of disease surveillance data for Europe: data sent to ECDC’sTESSy (The European Surveillance System) and other surveillance systems should be robust and backed by qualitylaboratory diagnostics.The handbook addresses the changing technological landscape that has emerged over the last decade, particularlywith regard to non-microbiological assays and platforms. Much of this technology has led to a major shift in TBdiagnostic activities with the development of multiple, large and well-equipped diagnostic centres with similarcapacity and skills to national reference laboratories (e.g. rapid molecular diagnostic tests). Conversely, othermolecular diagnostic developments have moved us away from ‘big laboratory’ approaches and closer to ‘point-ofcare’ devices. The two approaches are not mutually exclusive and both bring advanced diagnostics closer to thepatient. The handbook is designed to meet the needs of both centralised and decentralised service delivery modelsand recognises that the role of national reference laboratories will change significantly over the next few years.How this handbook relates to other work available in thisfieldThis handbook presents a compilation of methods currently applied in EU/EEA Member States. It describescommon work carried out and endorsed by European laboratory experts. It also features methods and proceduresdeveloped or refined by ERLTB-Net network partners.What this document is/is notThis document is a handbook of agreed methods in the field of TB diagnostics for laboratories serving referencefunctions in Europe. It provides a comprehensive compilation of key methods for the diagnosis of TB. Relevantstakeholders are encouraged to use this compilation as a basis for the validation, development, updating anddissemination of information.The current document does not contain any formal recommendations for implementation of specific methods inEU/EEA Member States. Recommendations and protocols contained within the handbook are not mandatory forEU/EEA laboratories.1

Handbook on TB laboratory diagnostic methods for the EUTECHNICAL DOCUMENTIntended use and usersThe report, which provides both basic- and reference-level methods for the diagnosis of TB, is intended for use bylaboratory experts. It will also be of interest to public health professionals in the field of global TB control,particularly those involved in European initiatives to foster progress towards elimination of TB.History of the handbookThe first annual meeting of the ERLN-TB was held in Stockholm in 2010. During this meeting, the network partnerscame to a consensus agreement about the relevant topics to be included in a handbook on (reference) laboratorymethods for the diagnosis of TB. The approach was to include several standardised and reliable methods, ratherthan to focus on one single method. A dedicated writing committee was formed to compile the first draft of thesemethods, using a handbook format. Each chapter includes descriptions of standardised diagnostic methods andhighlights key considerations regarding biosafety and quality assurance.The first edition was published in 2011. The handbook was extensively revised during 2014–2015 following theformation of the new ERLTB-Net network and in light of decisions made during its first annual meeting inSeptember 2014. A draft of the handbook was sent out to all ERLTB-Net network partners for review andendorsement in February 2015. An approved version was submitted to ECDC in March 2015.This editionThis handbook represents the second edition of a publication on the most reliable TB diagnostic methods,endorsed by the members of the ERLTB-Net. This publication is a compilation of protocols for the laboratorydiagnosis of TB, designed for laboratory experts and public health professionals. It was compiled to contribute tothe harmonisation of methods in the field of TB diagnosis in the EU/EEA, EU enlargement countries and EuropeanNeighbourhood Policy (ENP) partner countries, with the goal of ensuring comparability of TB diagnoses in Europe,and provision of the best care possible for TB patients, based on a quality-assured diagnosis. This publication canalso support laboratories in establishing a safe working environment for staff by minimising the risk of exposure toM. tuberculosis. The second edition of the ERLTB-Net handbook consists of ten chapters, each with a list ofrelevant references. Below is a summary of each chapter.1 Biosafety in clinical laboratory diagnosis of tuberculosisM. tuberculosis can cause laboratory-acquired infections. In order to ensure effective infection control, it is crucialthat a comprehensive and strict biosafety policy is developed and followed. Such a policy includes standardisedrules and regulations for containment, personal protective equipment (PPE), standard operating procedures (SOP)for different laboratory tasks and a transparent structure for regulating safe working conditions in diagnostic TBlaboratories.2 Quality assuranceNational tuberculosis programmes are supported by laboratories that provide reliable and quality-assured results.The chapter provides a comprehensive overview of existing International Organization for Standardization (ISO)standards relevant for the laboratory diagnosis of TB and describes internal and external quality assuranceprocedures.3 Latent TB infectionsCurrently, two types of tests are used for the diagnosis of latent TB infections (LTBI): tuberculin skin tests (TSTs)and Interferon-γ release assays (IGRAs). The chapter describes in detail the two commercially available IGRAs forthe detection of latent TB infection and provides support for interpreting and reporting test results.4 Smear microscopyTwo types of staining are most commonly used for the detection of mycobacteria: carbol-fuchsin staining (ZiehlNeelsen, Kinyoun) and fluorochrome staining (auramine, auramine-rhodamine). The chapter describes thepreparation of the required reagents and the sputum smear samples, as well as the staining procedures and thesystem foe reporting results.5 Culture for Mycobacterium tuberculosis complexThe use of cultures improves the sensitivity and specificity of TB tests, particularly at the early stages of thedisease, in cases of extrapulmonary tuberculosis (EPTB) and in the event of treatment failure. The chapter providesan overview of key principles for sampling and transporting clinical specimens and processing sputum and otherspecimens before inoculation to solid and liquid culture media, culture incubation and examination. The issue ofcontamination is addressed, along with the measures necessary to prevent laboratory-acquired TB infections.2

TECHNICAL DOCUMENTHandbook on TB laboratory diagnostic methods for the EU6 Identification of Mycobacterium tuberculosis and drug resistance incultures and sputum using molecular assaysGrowth of mycobacteria in culture media can take up to eight weeks as M. tuberculosis and many othermycobacteria grow very slowly. This can lead to a delay in the treatment of patients. Molecular assays can speedup mycobacterium identification and drug susceptibility testing, and thus lead to faster and more specifictreatment. The chapter describes methods for the identification of mycobacteria and drug susceptibility fromculture and clinical specimens and gives suggestions for the interpretation and reporting of results.7 First- and second-line drug susceptibility testing for Mycobacteriumtuberculosis complexThe main objectives of drug susceptibility testing are to improve individual treatment management of tuberculosiscases and drug-resistance surveillance at the level of a hospital, city, region, or country. The chapter describesthree different methods for drug susceptibility testing of first- and second-line drugs on solid and liquid media.8 Molecular typing of Mycobacterium tuberculosis complex isolatesVarious DNA fingerprinting methods are c

The aim of this handbook is to provide network members and other laboratories involved in the diagnosis of tuberculosis, with an agreed list of key diagnostic methods and their protocols in various areas of TB diagnosis, ranging from microbiological diagnosis of active TB to the diagnosis of latent TB infection (LTBI). This handbook