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TECHNICAL DOCUMENT

Handbook on TB laboratory

diagnostic methods in the European Union

ECDC TECHNICAL DOCUMENT

Handbook on TB laboratory diagnostic

methods for the European Union ii with support from Marieke J. van der Werf, Francis Drobniewski and Vladyslav Nikolayevskyy.

This report was sent for consultation to the members of the ERLTB-Net network (see Appendix 1 for list of

contributors).

The first version of this ECDC technical report, previously published as µMastering the basics of TB control:

handbook which was included as an annex. This report has now been revised, updated and renamed as the

Suggested citation: European Centre for Disease Prevention and Control. Handbook on TB laboratory diagnostic

methods for the European Union, Stockholm: ECDC; 2016.

Stockholm, March 2016

ISBN 978-92-9193-739-4

doi 10.2900/216384

Catalogue number TQ-01-16-109-EN-N

© European Centre for Disease Prevention and Control, 2016 Reproduction is authorised, provided the source is acknowledged Handbook on TB laboratory diagnostic methods for the EU TECHNICAL DOCUMENT iii

Contents

Abbreviations ................................................................................................................................................ v

Background and introduction .......................................................................................................................... 1

1 Biosafety in the laboratory diagnosis of tuberculosis ....................................................................................... 4

1.1 Background and principles ................................................................................................................... 4

1.2 The containment laboratory (biosafety level 3) ...................................................................................... 5

1.3 The laboratory facility .......................................................................................................................... 6

1.4 Procedures ......................................................................................................................................... 7

1.5 Personal protective equipment (PPE)..................................................................................................... 7

1.6 The human resource component .......................................................................................................... 8

1.7 Specific laboratory tasks related to hazards ........................................................................................... 8

2 Quality assurance ...................................................................................................................................... 12

2.1 Background and principles ................................................................................................................. 12

2.2 Selection and implementation of tests ................................................................................................. 13

2.3 Validation ......................................................................................................................................... 13

2.4 Control and trend monitoring by key indicators .................................................................................... 14

2.5 Physical and environmental conditions ................................................................................................ 14

2.6 Equipment ........................................................................................................................................ 14

2.7 Management and staff ....................................................................................................................... 14

2.8 Documentation and registration .......................................................................................................... 15

2.9 Sampling guidance, review of requests and service to customers ........................................................... 15

2.10 Subcontracting, services and supplies ................................................................................................ 15

2.11 Complaints, errors and corrective actions ........................................................................................... 15

2.12 Improvements................................................................................................................................. 15

2.13 Reporting and releasing results ......................................................................................................... 16

3. Diagnosis of latent TB infection ................................................................................................................. 17

3.1 Background and principles ................................................................................................................. 17

3.2 The tuberculin skin test (TST) ............................................................................................................ 18

3.3 Interferon-DŽ UHOHMVH MVVM\V H*5$V .................................................................................................... 18

4. Smear microscopy .................................................................................................................................... 32

4.1 Background and principles ................................................................................................................. 32

4.2 Procedure 1: Ziehl-Neelsen (ZN) ......................................................................................................... 32

4.3 Procedure 2: Fluorochrome staining .................................................................................................... 34

4.4 Sample collection .............................................................................................................................. 35

4.5 Smear preparation ............................................................................................................................ 36

4.6 Staining procedures ........................................................................................................................... 36

4.7 Microscopy ....................................................................................................................................... 37

4.8 Recording and reporting .................................................................................................................... 38

4.9 Quality control .................................................................................................................................. 40

5. Culture tests for Mycobacterium tuberculosis complex ................................................................................. 42

5.1 Background and principles ................................................................................................................. 42

5.2 Biohazards and biosafety in the TB laboratory ...................................................................................... 42

5.3 Specimen collection, storage and transport .......................................................................................... 44

5.4 Homogenisation and decontamination of specimens ............................................................................. 45

5.5 Culture media: principles ................................................................................................................... 49

5.6 Solid media ....................................................................................................................................... 49

5.7 Culture tube inoculation ..................................................................................................................... 50

5.8 Culture incubation ............................................................................................................................. 51

5.9 Culture examination .......................................................................................................................... 51

6. Identification of M. tuberculosis and drug resistance in cultures/sputum using molecular assays and immunoassays .. 54

6.1 Background and principles ................................................................................................................. 54

6.2 Procedure 1: Chromatographic immunoassay for the qualitative detection of Mycobacterium tuberculosis complex

from cultures ........................................................................................................................................... 54

6.3 Procedure 2: Line probe assay for identification of the genus Mycobacterium and 16 different mycobacterial

species including M. tuberculosis complex from culture .............................................................................. 55

6.4 Procedure 3: Line probe assay for identification of M. tuberculosis complex and detection of rifampicin resistance in M.

tuberculosis ............................................................................................................................................. 57

6.5 Procedure 4: Line probe assay for identifying species from genus Mycobacterium and detecting potential

MDR- and XDR-TB from cultures and smear-positive specimens .................................................................. 62

6.6 Procedure 5: Nucleic acid hybridisation test for the identification of M. tuberculosis complex .................... 69

6.7 Procedure 6: Single-use sample-processing cartridge system with integrated multicolour real-time PCR capacity for

detection & identification of M. tuberculosis complex bacteria and detection of rifampicin resistance ............................. 70

7. First- and second-line drug susceptibility testing for Mycobacterium tuberculosis complex ............................... 72

TECHNICAL REPORT Handbook on TB laboratory diagnostic methods for the EU iv

7.1 Background and principles ................................................................................................................. 72

7.2 Procedure 1: General method ± DST by culture .................................................................................... 72

7.4 Procedure 3: Resistance ratio method ................................................................................................. 76

7.5 Procedure 4: Drug susceptibility testing in liquid media (MGIT 960) ....................................................... 78

7.6 Procedure 5: Other methods for first-line drug susceptibility testing ....................................................... 79

8. Molecular typing of Mycobacterium tuberculosis complex isolates ................................................................. 83

8.1 Background and principles ................................................................................................................. 83

8.2 Procedure 1: Spoligotyping ................................................................................................................ 83

8.3 Procedure 2: MIRU-VNTR typing ......................................................................................................... 85

8.4 Procedure 3: RFLP typing ................................................................................................................... 86

8.5 General considerations on routine genotyping methods ........................................................................ 86

8.6 Procedure 4: whole genome sequencing ............................................................................................. 86

8.7 Materials .......................................................................................................................................... 87

8.8 Results/interpretation ........................................................................................................................ 88

8.9 Quality control .................................................................................................................................. 88

8.10 Biosafety ........................................................................................................................................ 88

9. Use and validation of disinfectants for Mycobacterium tuberculosis ............................................................... 92

9.1 Liquid disinfection ............................................................................................................................. 92

9.2 Gaseous disinfection .......................................................................................................................... 94

10. Information for physicians: laboratory diagnosis of tuberculosis ± first steps ................................................ 98

10.1 Introduction .................................................................................................................................... 98

10.2 General considerations regarding the diagnosis of tuberculosis ............................................................ 98

10.3 Specific considerations regarding diagnosis of tuberculosis .................................................................. 99

10.4 Information flow from microbiologist to physician and instructions regarding tuberculosis diagnosis ...... 100

Figures

Figure 1.1 Biohazard warning sign for laboratory doors ..................................................................................... 5

Figure 3.1 Potential outcomes of human host infection by M. tuberculosis ........................................................ 18

Figure 4.1 Microscope components ................................................................................................................ 37

Figure 4.2 AFB smear microscopy of biological specimens ................................................................................ 39

Figure 4.3 AFB smear microscopy of M. tuberculosis cultures .......................................................................... 39

Figure 5.1 Minimal examination schedule for solid cultures ............................................................................. 52

Figure 6.1 Examples of chromatographic immunoassays for qualitative detection of M. tuberculosis complex ..................... 55

Figure 6.2 Interpretation scheme for INNO-LiPA ............................................................................................. 57

Figure 6.3 Interpretation of LiPA ................................................................................................................... 61

Figure 6.4 Interpretation sheet for GenoType Mycobacterium CM ..................................................................... 65

Figure 6.5 Interpretation sheet for GenoType Mycobacterium AS ..................................................................... 66

Figure 6.6 Interpretation sheet for GenoType Mycobacterium MTBC ................................................................. 66

Figure 6.7 Interpretation sheet for GenoType Mycobacterium MTBDRPlus ......................................................... 67

Figure 6.8 Interpretation sheet for GenoType Mycobacterium MTBDRs ............................................................. 67

Tables

Table 1.1 Examples of personal protective equipment ...................................................................................... 8

Table 2.1 Overall scope of ISO 17025 and ISO 15189 .................................................................................... 12

Table 2.2 Central terms and abbreviations in quality assurance ....................................................................... 13

Table 3.1 List of national LTBI diagnosis guidelines ........................................................................................ 19

Table 3.2 QuantiFERON-TB Gold results interpretation .................................................................................... 24

Table 3.3 QuantiFERON-TB Gold PLUS results interpretation ........................................................................... 24

Table 3.4 Materials provided by the manufacturer .......................................................................................... 26

Table 4.1 Equipment required for smear preparation and staining .................................................................... 36

Table 4.2 Reporting of microscopy smears .................................................................................................... 38

Table 4.3 Common causes of error in smear microscopy ................................................................................. 40

Table 7.1 Solvents and diluents ..................................................................................................................... 74

Table 7.2 Calculation of modal MIC value ....................................................................................................... 76

Table 7.3 Examples of MIC interpretation ....................................................................................................... 77

Table 7.4 Critical concentrations of the most important first-line and second-/third-line drugs ............................ 78

Table 9.1 Differences between the two major gaseous hydrogen peroxide technologies, Steris VHP and Bioquell HPV .............. 95

Handbook on TB laboratory diagnostic methods for the EU TECHNICAL DOCUMENT v

Abbreviations

AFB Acid-fast bacilli

BCG Bacillus Calmette-Guérin

BD Becton and Dickinson

BSC Biological safety cabinet

BSL Biosafety level

CFU Colony forming units

CSF Cerebral spinal fluid

CRI Colorimetric redox indicator

CXR Chest X-ray

DNA Deoxyribonucleic acid

DST Drug susceptibility testing

EEA European Economic Area

ENP European Neighbourhood Policy

EPTB Extrapulmonary tuberculosis

ERLN-TB European Reference Laboratory Network for Tuberculosis (2010±2014) ERLTB-Net European Reference Laboratory Network for Tuberculosis (from 2014)

EQA External quality assessment

EU European Union

HEPA filter High-efficiency particulate air filter

HPF High-power field

IATA International Air Transport Association

IFN-Ȗ Interferon-gamma

INSTAND e.V. Society for promoting quality assurance in medical laboratories

IGRA Interferon-gamma release assay

IQA Internal quality assessment

ISO International Organization for Standardization

LED Light-emitting diode

LTBI Latent TB infection

MGIT Mycobacteria growth indicator tube

MIC Minimum inhibitory concentration

MIRU Mycobacterial interspersed repetitive units

MODS Microscopically observed drug susceptibility

MTBC Mycobacterium tuberculosis complex

NAAT Nucleic-acid amplification test

NRA Nitrate reductase assay

NRL National reference laboratory

NTM Non-tuberculous mycobacteria

PBMC Peripheral blood mononuclear cells

PCR Polymerase chain reaction

PPD Purified protein derivative

PPE Personal protective equipment

PPV Positive predictive value

QA Quality assurance

QC Quality control

RFLP Restriction fragment length polymorphism

RNI Reactive nitrogen intermediates

SDS Sodium dodecyl sulfate

SNP Single nucleotide polymorphism

SOP Standard operating procedures

TB Tuberculosis

TST Tuberculin skin test

UKAS United Kingdom Accreditation Service

UK NEQAS UKAS accredited proficiency testing provider No. 4715

VNTR Variable number tandem repeat

ZN Ziehl-Neelsen (staining method)

TECHNICAL DOCUMENT Handbook on TB laboratory diagnostic methods for the EU 1

Background and introduction

Francis Drobniewski

Tuberculosis (TB) is a major cause of morbidity and mortality in Europe. High-quality laboratory diagnosis of TB is

the basis for both individual patient treatment and surveillance.

In 2007, a survey of existing mycobacterial laboratory services and quality control practices throughout the EU

confirmed the key role of national reference laboratories (NRL) for TB and their services. The main conclusion of

the survey was that a network of reference laboratories for tuberculosis could contribute to improving the

performance of mycobacterial laboratories in Europe.

Based on these results, the European Reference Laboratory Network for Tuberculosis (ERLN-TB) was launched in

January 2010, with the aim of strengthening TB diagnostics in the European Union. ERLN-TB was funded and

coordinated by ECDC. One or two officially nominated reference laboratories from each Member State joined the

network, 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 called

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