GLASS

Guidance for national

reference laboratories

Global Antimicrobial Resistance and

Use Surveillance System (GLASS)
GLASS
Guidance for national
reference laboratories

Global Antimicrobial Resistance and

Use Surveillance System (GLASS)
GLASS guidance for national reference laboratories
ISBN 978-92-4-001058-1 (electronic version)
ISBN 978-92-4-001059-8 (print version)
© World Health Organization 2020
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Contents

Acknowledgements iv
Acronyms v
Preface vi
1 National reference laboratories for surveillance
of antimicrobial resistance 1

2 Functions of a national reference laboratory in

a network of national surveillance laboratories 3

3 Reference functions 4
3.1 Reference of samples or isolation 5
3.2 Confirmation and characterization of resistance mechanisms 6
3.3 Quality control for surveillance sites that support clinical laboratories 10
3.3.1 Indirect quality control 10
3.3.2 Quality control system 11
3.3.3 External quality assessment programme 12
3.4 Outbreak support 13

4 Guidance and standardization 14

5 Training 15

6 Data collection and analysis 16

6.1 Laboratory information systems and test reports 16
6.2 Data analysis for national surveillance of antimicrobial resistance 17

7 Assessment of laboratories 18

8 References 19

iii
Acknowledgements

WHO thanks the following: Contributions by staff at WHO regional offices

Reviewers at WHO collaborating centres Sheick Oumar Coulibaly and Laetitia Gahimbare, Regional
Office for Africa; Nienke Bruinsma, Marcelo Galas and
Rene S. Hendriksen (WHO Collaborating Centre, National Ramon Pardo Pilar, Regional Office for the Americas; Walaa
Food Institute, Technical University of Denmark); Monica Khater, Regional Office for the Eastern Mediterranean; Danilo
Lahra (WHO Collaborating Centre for Sexually Transmitted Lo Fo Wong, Regional Office for Europe; and Aparna Singh
Infections and Antimicrobial Resistance, Australia); Jean Shah and Sirenda Vong, Anuj Sharma, Regional Office for
Patel (WHO Collaborating Centre, Centers for Disease South-East Asia.
Control and Prevention, USA); Wantana Paveenkittiporn
(WHO Collaborating Centre for Community Nutrition and WHO staff at headquarters
Food Safety, Thailand); Olga Perovic (WHO Collaborating
Centre for Antimicrobial Resistance, National Institute for Jorge Raul Matheu Alvarez, Sebastien Cognat, Sergey
Communicable Diseases, South Africa); and Neil Woodford, Romualdovich Eremin, Sapna Manglani, Christopher
Nandini Shetty, Katie Hopkins (WHO Collaborating Centre Oxenford and Carmem Lucia Pessoa-Silva.
for Reference and Research on Antimicrobial Resistance and
Healthcare Associated Infections, United Kingdom). Developer group

External reviewers Jorge Raul Matheu Alvarez, Sapna Manglani, Jean Patel and
Carmem Lucia Pessoa-Silva.
Alejandra Corso (Laboratorio Nacional de Referencia en
Resistencia a los Antimicrobianos, Ministry of Health, Executive group
Argentina), Runa Jha (National Public Health Laboratory,
Nepal), Onur Karatuna (European Committee on Antimicrobial Jorge Raul Matheu Alvarez and Carmem Lucia Pessoa-Silva.
Susceptibility Testing Development Laboratory, Sweden)
and Norio Ohmagari (National Center for Global Health and Financial support
Medicine Hospital, Japan).
The Government of the Republic of Korea through the Korea
International Cooperation Agency and the Government of the
United States of America.

iv
Acronyms

antimicrobial resistance
AMR
antimicrobial susceptibility testing
AST
Clinical and Laboratory Standards Institute
CLSI
external quality assurance
EQA
European Committee on Antimicrobial Susceptibility Testing
EUCAST
Global Antimicrobial Resistance and Use Surveillance System
GLASS
national reference laboratory
NRL
PCR polymerase chain reaction
quality control
QC

v
Preface

Antimicrobial resistance (AMR) is a significant, increasing threat to global public health.

In 2015, WHO launched the Global Antimicrobial Resistance and Use Surveillance
System (GLASS) for standardized collection of data on AMR at the global level to inform
policy to monitor the effectiveness of interventions and strengthen the evidence base
on AMR. Surveillance of antimicrobial resistance requires reliable microbiological
analysis of clinical specimens, and national reference laboratories (NRLs) play a key role
in supporting AMR surveillance in countries. Upon enrolment in GLASS, countries are
requested to designate at least one NRL with expertise in methods for characterizing
AMR pathogens and providing support to the national AMR surveillance system.
Important functions of NRLs include the provision of guidance and support to clinical
laboratories in the surveillance system for adopting national standards and protocols for
microbiological analysis. The NRL works in tandem with the national AMR surveillance
coordinating centre to define strategies for AMR surveillance. The NRL also plays a key
role in confirmation and timely reporting of emerging resistance to allow prompt and
effective prevention and control.
NRLs are a central part of national surveillance systems in countries, with a key role
in public health and in the diagnosis of a range of diseases. This technical guidance
focuses specifically on the functions and activities of NRLs for national surveillance
of AMR. Details of the various functions are provided, including reference functions
such as confirmation and characterization of resistance mechanisms, quality control
for surveillance sites, external quality assessment, outbreak support, guidance and
standardization, test validation and verification, providing training, data collection and
analysis for national surveillance of AMR and laboratory assessments.
Countries can use this technical guidance to establish or improve NRL capacity within
the national AMR surveillance system. Increased laboratory capacity in countries and
rapid, accurate diagnostic testing will significantly strengthen global AMR surveillance
and diagnostic stewardship.
WHO is grateful for the support of the international WHO AMR Surveillance and Quality
Assessment Collaborating Centres Network for the development of this technical note.

vi
 GLASS guidance for national reference laboratories

01
National reference
laboratories for surveillance
of antimicrobial resistance

National reference laboratories (NRLs) are a key component of national surveillance

systems for diagnosis of disease in public health. While NRLs are essential for all
microbiological diagnosis and special analyses in areas such as in virology, parasitology,
mycology and bacteriology, this document addresses the role, functions and activities
of an NRL in national surveillance for antimicrobial resistance (AMR). It can be used as
guidance for countries to ensure that their surveillance systems comply with the WHO
Global Antimicrobial Resistance and Use Surveillance System (GLASS).
The GLASS Manual for Early Implementation (1) calls for at least one national laboratory in
each WHO Member State to be designated as the NRL for AMR surveillance. The designated
laboratory must have expertise in methods for characterizing AMR-resistant pathogens.
The overall role of the NRL is to promote and facilitate good laboratory practice in the
country and promote harmonization of the methods and standards used in AMR national
surveillance systems, such as the guidelines of the European Committee on Antimicrobial
Susceptibility Testing (EUCAST) and of the Clinical and Laboratory Standards Institute
(CLSI). These standards should be met in all national AMR surveillance systems, in line with
the recommendations in the GLASS Manual (2) and according to the principles and practice
of general bacteriology. In addition to supporting the laboratory network, the NRL should
liaise with the national coordinating centre to define strategies for AMR surveillance and, in
particular, microbiological indicators, facilitate capacitation of laboratories at surveillance
sites and assist the national coordinating centre in implementing strategies to ensure that
microbiological information is combined with clinical and epidemiological data. The NRL
has a key role in confirming and reporting emerging resistance, in accordance with the
GLASS framework for reporting emerging antimicrobial resistance (3) (Fig. 1).

1
GLASS guidance for national reference laboratories

Fig. 1. National reference laboratory supports laboratories at surveillance sites

Surveillance sites Surveillance sites Surveillance sites

National
Reference
Laboratory

In the absence of national capacity for NRL functions, international collaboration can
be established ad interim with regional or international institutions. The WHO AMR
Surveillance and Quality Assessment Collaborating Centres Network (4) was established
to support countries, and particularly low-income countries, to build capacity for AMR
surveillance. The Network can assist NRLs in capacity-building, confirmatory testing and
further characterization if necessary.
The NRL coordinates, facilitates and guides laboratories at surveillance sites in a
national AMR surveillance network. The NRL promotes good laboratory practice and
standardization and harmonization of laboratory protocols and ensures their application
in surveillance laboratories. The NRL should organize or facilitate the participation of all
laboratories at surveillance sites in national external quality assurance (EQA) schemes to
monitor performance, to ensure gradual improvement of the laboratories’ work and the
reliability of their data. Transparent discussion of national EQA results will encourage
laboratories to improve performance and processes. The NRL must have the capacity and
capability to perform confirmatory testing, such as verification of species identification and
antimicrobial susceptibility testing (AST), including determination of minimum inhibitory
concentrations. The NRL should validate microbiological data provided by local surveillance
sites in collaboration with the national coordinating centre. Ideally, the NRL should have the
capacity to perform or have access to special tests, such as molecular testing and genome
sequencing, for characterizing resistance mechanisms, including use of bioinformatics to
analyse genomic data.
NRLs are encouraged to participate in international workshops on AST and molecular
methods for AMR, including new technologies such as whole-genome sequencing, for
analysing and characterizing determinants of resistance. Such workshops can provide
training and sharing of experience among countries. NRL staff can also benefit from
exchanges and training at NRLs in other countries with well-established national AMR
surveillance networks or other centres of excellence in the field of AMR.

2
 GLASS guidance for national reference laboratories

02 Functions of a national
reference laboratory in
a network of national
surveillance laboratories

The NRL provides guidance and support to laboratories in surveillance systems to adopt
national standards and protocols and improve local, regional and national capability for
AMR surveillance. The NRL should develop or adopt protocols and standard operating
procedures to standardize the AMR methods to be used in the laboratories in the
surveillance system (Fig. 2). GLASS proposes international standards (to be adapted
locally) to ensure uniform global AMR monitoring (5, 6).

Fig. 2. NRL functions in support of laboratories in an AMR surveillance system

National reference
laboratory

Guidance and Data collection Evaluation

Reference Training Research
standardization and analysis visits

3
GLASS guidance for national reference laboratories

03 Reference functions

The NRL should provide reference testing to all laboratories at surveillance sites and
other laboratories that isolate, detect and identify bacterial species and confirm and
characterize AMR mechanisms.
An NRL has four reference functions:
• primary analysis of samples (when necessary); specialized and confirmatory testing
for identification; serotyping by analysing samples and identifying species, including
subtyping;
• confirmation and characterization of AMR mechanisms, including analyses that
cannot be performed at surveillance sites;
• outbreak investigation; and
• quality control (QC) and quality assurance.

4
 03 Reference functions

3.1 Reference of The NRL should provide support to surveillance sites for samples that require specialized
samples or isolation testing or at which the local laboratory has no testing capacity. An example is isolation of
Streptococcus pneumoniae, for which some laboratories have neither the capability nor the
capacity. Table 1 lists the support that an NRL can provide to surveillance sites.

Table 1. Submission of samples and isolates to an NRL for isolation, identification and characterization

SURVEILLANCE SITE SURVEILLANCE SITE NRL ACTIVITY CRITERIA FOR SHIPPING TO NRL
CAPACITY ACTIVITY

No capacity to Sample shipment: Bacterial Culture, isolation, Bacteria that cause pneumonia or meningitis
isolate fastidious infection suspected but no identification, AST N. gonorrhoeae
bacteria or inadequate laboratory and characterization
No capacity in local laboratory
capacity
(e.g. Salmonella serotyping)

Capacity to isolate Bacterial isolation, shipping Identification, AST The percentage of isolates to be characterized
and identify genus of isolate to NRL and characterization and confirmed should be determined according
but not to identify or to the resources of the NRL.
characterize species

Capacity to isolate Bacterial isolation, Sub-typing for Alla isolates should be sent for etiology-specific
and identify genus identification, shipping characterization surveillance, e.g. for bacterial meningitis and
and species but not isolate to NRL for pneumonia, foodborne diseases, diarrhoeal
to subtype or analyse characterization, syndromes
e.g. serotyping

Isolation and identification Confirmation and All the following GLASS pathogens: Salmonella
of fastidious or rare species characterization, spp., Shigella spp., S. pneumoniae, Neisseria
molecular testing, gonorrhoeae.
including sequencing Others based on capability of NRL (e.g.
of isolates Campylobacter spp., N. meningitidis,
Haemophilus influenzae).

a
During early implementation, few bacteria (e.g. Salmonella spp., N. gonorrhoeae) may be isolated. As the capacity and number of isolates increases
at surveillance sites, the percentage of isolates to be submitted and characterized by the NRL should be evaluated.

5
GLASS guidance for national reference laboratories

3.2 Confirmation When possible, the NRL should support surveillance sites in confirming or characterizing
and characterization major resistance mechanisms of species of national or international importance. The
of resistance identity of any isolate submitted to the NRL should be confirmed, especially if there is any
mechanisms doubt about the provisional identification from the submitting laboratory. The NRL should
use internationally recognized methods for accurate identification of a wide range of
microorganisms.
The NRL should provide confirmatory genotypic testing to verify any unusual resistance
reported by a surveillance site and test extensively drug-resistant pathogens for
susceptibility to an extended list of antimicrobials for which testing may not be available
in health care laboratories or surveillance sites. Table 2 lists the criteria for submitting
isolates for confirmatory testing to an NRL.

Table 2. Criteria for submitting isolates to an NRL for confirmation of resistance mechanisms

PATHOGEN PURPOSE OF TESTING AT THE NRLa

Klebsiella spp. and Confirmation of resistance or AST for an extended list of drugs when treatment options are
Escherichia coli isolates sent limited because of resistance
to NRL for AMR confirmation Phenotypic and genotypic characterization of AMR mechanisms, particularly those that confer
and characterization resistance to carbapenem, extended-spectrum cephalosporin or colistin, and others such as
fluoroquinolone or macrolides, according to local epidemiology (see Table 4 in section 3.3.1).
Phenotypic and genotypic characterization of multidrug-resistantb, extensively drug-resistantc
and pan-drug-resistantd isolates, according to local epidemiology (see Table 4).
Molecular typing or sequence analysis of isolates related to multidrug-resistant outbreak.

Acinetobacter spp. Confirmation of resistance or AST to an extended list of antimicrobials when treatment options
are limited because of resistance
Characterization of carbapenem-resistant isolates due to production of carbapenemases
(e.g. New Delhi metallo-β-lactamase, K. pneumoniae carbapenemase, Verona integron-encoded
metallo-β-lactamase, imipenemase metallo-β-lactamase), colistin resistance
Molecular typing of isolates related to outbreak investigations

Salmonella and Shigella spp. Susceptibility testing of all isolates or of a convenient set when susceptibility testing is not
routinely performed for patient care. Results are used to guide empiric treatment decisions and
to assess the potential impact of antimicrobial use in food-producing animals on human health.
Confirmation of resistance to new drugs or AST to an extended list of drugs when treatment
options are limited because of resistance
Characterization of resistance mechanisms that confer resistance to carbapenem or extended-
spectrum cephalosporin.
Epidemiological typing, including serotyping and molecular typing, of all isolates to detect
outbreaks or submission of selected isolates for typing during an outbreak investigation

Staphylococcus aureus Confirmation of resistance to new drugs or susceptibility testing to an extended list of drugs
when treatment options are limited because of resistance
Confirmation of resistance or decreased susceptibility to vancomycin
Confirmation of resistance to oxazolidinone (linezolid, tedizolid) and lack of susceptibility to
daptomycin or lipoglycopeptides (dalbavancin, oritavancin and telavancin)

6
 03 Reference functions

PATHOGEN PURPOSE OF TESTING AT THE NRLa

S. pneumoniae Confirmation of unusual resistance (e.g. to vancomycin or extended-spectrum cephalosporins)

or resistance to new drugs
Susceptibility testing to an extended list of drugs when treatment options are limited because of
resistance
Serotyping and molecular typing to identify vaccine escape strains

N. gonorrhoeae Susceptibility testing of alle isolates or of a convenient set when susceptibility testing is not
routinely performed for patient care. Results are used to guide empiric treatment decisions.
Confirmation of unusual resistance (e.g. to extended-spectrum cephalosporins or azithromycin)
or resistance to new drugs
Susceptibility testing to an extended list of drugs when treatment options are limited because
of resistance
Molecular typing or sequence analysis of isolates

a
 he number of isolates and resistance mechanisms to be sent to the NRL should be defined according to local resources, the prevalence of
T
the resistance mechanism and capacity but should allow for characterization of emerging and local resistance mechanisms to determine their
epidemiology.
b
Non-susceptibility to at least one agent in three or more antimicrobial categories (7)
c
Non-susceptibility to at least one agent in all but two or fewer antimicrobial categories (7)
d
Non-susceptibility to all agents in all antimicrobial categories (7)
e
There may be only a few isolates of N. gonorrhoeae in countries during early implementation. As the capacity and number of isolates increases at
surveillance sites, the percentage of isolates to be submitted and characterized by the NRL should be evaluated.

When possible, the NRL should use AST methods that complement and do not duplicate
the methods already used in the submitting laboratory. For example, if the submitting
laboratory usually uses disc diffusion testing, the NRL should further determine the
minimum inhibitory concentration and include a wide range of antimicrobials with
a test that is commercially available, such as a commercial automated system, broth
microdilution or agar dilution.
The mechanism of AMR, especially those that are likely to be transferrable, such as
plasmid-mediated resistance determinants, can provide important epidemiological
information on transmission to facilitate control and prevention of further spread.
For example, some new compounds for treating infections caused by carbapenem-
resistant Enterobacterales are active against some types but not others.
These Enterobacterales can produce various carbapenemases (e.g. K. pneumoniae
carbapenemase, New Delhi metallo-β-lactamase, oxacillinase-type β-lactamase-
48-like, imipenemase metallo-β-lactamase or Verona integron-encoded metallo-β-
lactamase). Identification of the most prevalent carbapenemases in a country or region
is useful for predicting drug activity and for guiding treatment and prevention. For
example, at present, there are few licensed treatments for New Delhi metallo-β-
lactamase-producing Enterobacterales; therefore, detection of isolates with this resistance
mechanism can indicate where action to prevent spread is necessary and whether new
treatments should be used.

7
GLASS guidance for national reference laboratories

Both phenotypic and genotypic tests should be used to confirm a resistance mechanism
such as β-lactamase production, because any discrepancy between results can indicate
new or emerging mechanisms of resistance. If an isolate is phenotypically resistant to
carbapenems, but molecular or genotypic characterization indicates that it is negative
for the most common carbapenemase-encoding genes, the isolate may have developed
resistance (e.g. acquired genetic mutations) or may harbour a new or uncommon resistance
mechanism gene. If only molecular or genotypic testing is performed, isolates with new or
unusual resistance genes will be missed.
Both commercial and non-proprietary tests are available for identifying resistance
mechanisms (8). The test(s) chosen should have good performance characteristics (i.e.
high specificity and sensitivity for the specimen or isolate tested), be quality assured by the
manufacturer, identify the most common resistance mechanisms and suit the capability
of the NRL. Polymerase chain reaction (PCR) is an economical, easy test for targeted
detection of resistance mechanisms. Whole-genome sequencing (9) can be used for more
comprehensive analysis of resistance mechanisms, with bioinformatics programs for
analysis. Several analytical tools can be used to identify resistance mechanisms. The tiers of
testing capability suggested for NRLs are listed in Table 3. Tier 1 tests are those that an NRL
should be able to perform routinely. An NRL should perform tier 2 tests or identify another
laboratory in which these tests can be done. Tier 3 tests require advanced testing capability.
The GLASS tool for reporting emerging AMR (10) provides guidance for NRLs in the
detection, confirmation, characterization and notification of emerging resistance.

Table 3. Capability of an NRL to support surveillance sites

PATHOGEN TIERa TEST PURPOSE OF TESTING RECOMMENDED ASSAYS AND

PLATFORMS
All (see N. gonorrhoeae -specific recommendations below)

1 Bacterial culture Identify infectious agent and Conventional culture methods

from clinical recover infectious isolates for Selective media for specific pathogens
specimens characterization

1 Bacterial Confirm identity of isolates submitted Biochemical tests to identify species

identification to NRL (manual or automated)

1 Susceptibility to Confirmation of antimicrobial Determination of minimal inhibitory

all drugs used for susceptibility and extended concentration (broth microdilution,
treatment drug testing. See below for agar dilution, gradient diffusion strips)
recommendations of drugs to be according to method recommended
tested. and standard criteria (EUCAST, CLSI)
Disc diffusion testing for QC of results
Automated methods

2b Molecular testing Detect and confirm resistance Conventional PCR, real-time PCR
mechanisms (commercial methods)

3 Whole-genome Sequencing isolates, strain typing, Next-generation sequencing platforms

sequencing detection of resistance or virulence,
characterization of transmission
dynamics during outbreak
investigations

a
 ier 1, minimal testing capability. Tier 2, preferred testing capability. NRLs should either perform this test or identify another laboratory that can
T
perform such testing when necessary. Tier 3, advanced testing capability.
b
Laboratories in tier 2 should perform tests for tiers 1 and 2. Laboratories in tier 3 should also perform tests for tiers 1 and 2.

8
 03 Reference functions

PATHOGEN TIERa TEST PURPOSE OF TESTING RECOMMENDED ASSAYS AND

PLATFORMS
Klebsiella spp. and E. coli

1 Phenotypic Detection of a transferrable Modified carbapenem inactivation test

carbapenemase test carbapenem resistance mechanism (could be performed in conjunction
with eCIM), CarbaNP

2 Molecular detection Identification of transferrable PCR testing (commercial and non-

of carbapenemases, resistance mechanisms proprietary tests available)
cephalosporinases Lateral flow immunoassay
and/or mcr-1 gene (commercial test available for
for plasmid-mediated carbapenemases and mcr gene)
colistin resistance
Acinetobacter spp.

2 Phenotypic Detection of a transferrable PCR testing (commercial and non-

carbapenemase test carbapenem resistance mechanism proprietary tests available)
(e.g. K. pneumoniae carbapenemase,
oxacillinase-type β-lactamase,
New Delhi metallo-β-lactamase) in
Acinetobacter spp.

2 Molecular detection Identification of transferrable

of Enterobacterales- resistance mechanisms
like carbapenemases
(e.g. K. pneumoniae
carbapenemase,
New Delhi metallo-
β-lactamase,
oxacillinase-type
β-lactamase)
Salmonella and Shigella spp.

1 Phenotypic Detection of a transferrable Extended-spectrum β-lactamase

extended-spectrum resistance mechanism inhibitor tests (see CLSI and EUCAST
β-lactamase and recommendations)
carbapenemase test Modified carbapenem inactivation test
(could be performed in conjunction
with eCIM), CarbaNP

2 Molecular detection Identification of transferrable PCR testing (commercial and non-

of carbapenemases, resistance mechanisms proprietary tests available)
cephalosporinases Lateral flow immunoassay
and/or mcr-1 gene (commercial test available for
carbapenemases and mcr gene)

2 Conventional Identification of outbreak strains Slide agglutination assay

serotyping

9
GLASS guidance for national reference laboratories

PATHOGEN TIERa TEST PURPOSE OF TESTING RECOMMENDED ASSAYS AND

PLATFORMS

vanA detection
Staphylococcus aureus

2 Identify the most likely resistance PCR testing

Vancomycin mechanism in isolates that are
Intermediate resistant to vancomycin. Consider
Staphylococcus testing for other van genes in isolates
aureus strains that are resistant to vancomycin but
vanA negative.
DAP-NS strains
LNZ-R strains

2 Detection of mecA Confirm resistance, and identify mecA and mecC PCR
and mecC the mechanism for epidemiological Protein-binding protein 2a latex
purposes agglutination
S.
pneumoniae

2 Conventional Detection of vaccine escape Quellung or latex agglutination

serotyping serotypes serotyping

3 PCR or sequencing to Alternative to serotyping PCR testing (commercial and

determine M type non-proprietary tests available)
N. gonorrhoeae

2 Bacterial culture Diagnosis of the infectious agent and Conventional culture methods.
from clinical recovery of infectious isolates for Selective media for specific pathogens.
specimens characterization

2 Testing of Detection of antimicrobial Agar dilution, disc diffusion or gradient

susceptibility to susceptibility where culture- diffusion strips
all drugs used for independent diagnostics are
treatment commonly used
Extended drug susceptibility testing

3 Whole-genome Isolate identification, molecular Next-generation sequencing platforms

sequencing typing, molecular detection of
resistance

3.3 Quality control 3.3.1 Indirect quality control

for surveillance sites An NRL should establish a programme to analyse and verify the results of monitoring of
that support clinical surveillance sites that support clinical laboratories. An indirect QC programme should be
laboratories established to receive isolates under surveillance with specific resistance mechanism(s)
to be confirmed by the NRL. The scheme should be established according to the NRL
resources and capacity to analyse isolates sent from surveillance sites, adapted to the
pathogens to be confirmed and characterized (section 3.2). The scheme is based on the
list of pathogens under surveillance and the main resistance mechanism(s) to be detected
at the surveillance sites. According to the prevalence of the pathogen or resistance
mechanism, a proportion of each combination of isolates is sent for QC and confirmation
of the resistance mechanism. Table 4 shows the scheme for QC and confirmation of
resistance mechanisms, including proportion and periodicity.

10
 03 Reference functions

Table 4. Scheme proposed for sending isolates from surveillance sites to NRLs for confirmation or characterization

PATHOGEN RESISTANCE MECHANISM PROPORTION OF ISOLATES TO BE SENT TO NRL

K. pneumoniae Extended-spectrum β-lactamase High prevalence, 10%

E. coli Low prevalence, ≤ 50%
Any other Enterobacterales
Carbapenem resistance 100%

Colistin resistance 100%

Acinetobacter baumannii Carbapenem resistance 10% should be established according to local

prevalence

Colistin resistance 100%

S. aureus Vancomycin intermediate S. aureus, 100%

vancomycin-resistant S. aureus, linezolid
resistance and non-susceptibility to
daptomycina

S. pneumoniae Vancomycina and extended-spectrum 100%

cephalosporin resistance, high-level
penicillin resistance

Salmonella spp. Extended-spectrum β-lactamase, 100%

Shigella spp. carbapenem resistance

Colistin resistance 100%

N. gonorrhoeae Third-generation cephalosporin resistance 100%

and azithromycin resistance

a
If these antimicrobials are tested at surveillance sites

3.3.2 Quality control system

All laboratories should control the quality of all tests performed, including AST. QC should
be performed and recorded to ensure that the results of all analyses are accurate and the
information is valid for use in clinical decisions and for broader local and national decisions
as part of the national surveillance system.
The NRL should guide clinical laboratories in establishing internal QC schemes and provide
guidance in the development and use of standard operating procedures for bacteriology
and AST. Internal QC testing schemes should be evaluated by the NRL during visits to
evaluate the performance of the laboratories participating in the system. GLASS tools are
available to assess the performance of surveillance sites (11).
In countries with a laboratory regulatory authority, the authority may recommend testing
practices. Many laboratories have individual QC plans rather than a defined QC programme.
For AST, information on QC strains and the expected ranges of test results are available in
CLSI document M100 (5) and EUCAST QC tables for AST of bacteria (6). QC strains can be
obtained from culture collections, such as the American type culture collection (12) or the
United Kingdom national collection of type cultures (13).

11
GLASS guidance for national reference laboratories

3.3.3 External quality assessment programme

For all routine tests, which may also be on clinical samples, the NRL should participate in
an EQA programme as a part of the quality assurance system. NRLs should also establish
a national EQA programme for laboratories participating in the AMR surveillance system.
EQA testing should be adapted to national resources and capacity, but specimens for
AMR EQA should be distributed to all clinical laboratories participating in the national AMR
surveillance system at least once a year, with six to eight unknown strains to assess testing
of the main target pathogens and resistance mechanisms, or three to four strains per round
twice a year. An EQA programme determines the ability of laboratories to detect major
resistance mechanisms and unusual and emerging resistance and to perform specific tests
for bacterial identification. The EQA results should be used to improve testing capacity and
capability locally or more widely, with assessment visits, training, updating protocols and
monitoring performance.
WHO supports EQA programmes for bacterial identification and AST in several regions.
The minimal requirements for regional EQA programmes for NRLs that support AMR
surveillance are listed in Table 5.

Table 5. Minimal requirements for regional EQA programmes

MINIMAL TECHNICAL REQUIREMENTS ACCEPTABLE MINIMAL REQUIREMENTS

Clinical information included for each specimen sample or Minimal clinical information included for each specimen
isolate sample or isolate (e.g. specimen type, diagnosis)

Target pathogens GLASS pathogens should be the main focus at initial stages
and others included as considered necessary in each EQA
programme

Antibiotics tested for susceptibility and resistance, Panel of antimicrobials considered necessary to test the
antibiogram capacity of the NRL to detect and confirm the main resistance
mechanism by species

AST standard used: EUCAST or CLSI Providers should be able to perform and analyse both in order
to test NRLs that use different standards.

AST method used: laboratory screening method (e.g. disc Providers should be able to perform any AST method,
diffusion, minimum inhibitory concentration, epsilometric test) according to the adopted guidelines. NRL participants should
state the method used (e.g. disc diffusion, automated system).

Total number of samples or isolates per panel At least 5–6 isolates per panel

Transport method (freeze dried, agar in cryotube) Method up to the provider (lyophilized preferred), following
standards (e.g. certain specimen types must be shipped
rapidly).

Methods (including reagent brands) requested for EQA programmes should know the methods used by NRLs for
identification bacteria identification. A detailed description of the method is
essential to identify areas for improvement if the NRL does not
perform to standard.

AST result requested EQA programmes should ask the NRL to include the results
Interpretation, zone diameter, minimum inhibitory for the method in millimetres or concentration and its
concentration interpretation.

AST interpretation errors: minor, major, very major EQA programmes should analyse and report the types of errors
found for each result, isolate and participating laboratory.

Additional characterization requested (serotyping, AST EQA programmes should ask the NRL for confirmation of
confirmatory test) resistance mechanisms and serotyping of species included in
the panel (e.g. Salmonella, extended-spectrum β-lactamase,
Klebsiella pneumoniae carbapenemase)

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 03 Reference functions

ORGANIZATION ACCEPTABLE MINIMAL REQUIREMENT

Provider: accreditation is desirable. Laboratory accreditation of the provider is desirable.

Provider should participate in an international EQA for providers.

Target participants of regional EQA programmes For regional EQA programmes, NRLs

Frequency of EQA panels per year At least once a year

ANALYSIS AND FEEDBACK ACCEPTABLE MINIMAL REQUIREMENT

Post-EQA follow-up: training, on site visits, etc. Ideally, the provider should write a report and guidance for
rectifying the main issues identified, according to species or
resistance mechanism.

EQA software Ideally, the provider should have software or a specific electronic
template to facilitate the reporting and analysis of results.

SHIPPING OF EQA PANEL ACCEPTABLE MINIMAL REQUIREMENT

Shipping and logistical challenges Provider should ensure and use international standards for
shipping and transport of biological agents.

EQA FOLLOW UP ACTIVITIES ACCEPTABLE REQUIREMENT

Training and assessment visits Based on the results of the Regional EQA, labs should consider
a training program and assessement visit to NRL to improve
capacity and performance.

3.4 Outbreak support The NRL should support and increase local testing of isolates during an outbreak
investigation, working with an infection prevention and control committee or infectious
diseases programme in health facilities. Testing should be complemented by an
epidemiological investigation of the outbreak to identify measures to stop transmission
of the outbreak pathogen. If necessary, samples or isolates collected from cultures in
hospitals, on site or in mobile laboratories may be submitted to the NRL and tested to
confirm the isolate, the susceptibility profile, the mechanism of resistance and, if the
NRL has the capacity, the dynamics and sources of transmission by molecular testing of
isolates. If resistant bacteria in an outbreak require laboratory testing that the NRL cannot
provide, NRL staff may seek assistance from the WHO AMR Surveillance and Quality
Assessment Collaborating Centres Network (4) or from another international institution
and, when possible, from institutions that are regional reference laboratories for WHO
regional offices.
For some investigations, it may be necessary to identify patients who are colonized with the
resistant pathogen. In this case, tests (e.g. culture or PCR) may be performed in a hospital
laboratory or the NRL, according to the availability of resources.

13
GLASS guidance for national reference laboratories

04 Guidance and
standardization

As national coordinators of laboratory activities in the AMR surveillance system,

NRLs should establish the standards to be used. The two most common international
standards are those of EUCAST and CLSI, both of which include the methods and
breakpoints to be used. The NRL should carefully consider the resources necessary
to implement any standard sustainably. It should consult the national antibiotic
committee, if such a body exists, to ensure consistency and advocate use of the agreed
standard throughout the national AMR surveillance network.
Other national guidance to be developed and distributed by NRLs should include:
• guidance on laboratory activities to support the AMR surveillance system;
• standard operating procedures for the samples and pathogens under surveillance;
• internal quality control procedures;
• participation in national EQA and monitoring of performance;
• guidance for collection, transport and storage of samples and referral of isolates,
including a flowchart for shipping;
• guidance on standardized procurement of laboratory supplies; and
• guidance on data collation, analysis and development of antibiograms.
The performance characteristics of all testing methods should be determined before
they are used, which is called “validation” if the method was developed in house.
Validation consists of comparing the results obtained in using the method with those
of a reference method or a “gold standard” on a group of samples or isolates that are
representative of the population. Method validation usually includes comparison of the
sensitivity and specificity of the method with those of a reference method. For AST, test
performance is evaluated by measuring the proportion of results that show essential
agreement (minimum inhibitory concentrations in a single doubling dilution) and the
proportion that show categorical agreement (results in the same interpretive category,
e.g. susceptible or resistant). The criterion for acceptance should be a performance
agreement ≥ 95%.
Every commercial test should be verified in every laboratory before it is introduced into
routine use, to confirm all relevant aspects of the manufacturers’ product performance
and to ensure that the tests are suitable for their intended use. Verification is similar to
validation, but a smaller sample of specimens or isolates is tested (e.g. with a panel of
QC strains). Verification should be conducted for every new product batch.

14
 GLASS guidance for national reference laboratories

05
Training

NRLs should develop a training programme for staff at the clinical laboratories that
support the surveillance sites, which should include:
• sample collection, transport and storage;
• bacterial isolation and identification;
• AST methods;
• resistance mechanisms, screening and confirmation;
• AMR surveillance and data analysis; and
• quality management and improvement.
Participation of all staff in training should be mandatory in order for the clinical
laboratory to be part of the surveillance system. NRLs should monitor the performance
of laboratories at surveillance sites in an EQA scheme, by assessing the correlation
between the results and the isolates sent, conducting visits and sharing the quality of the
data with the national surveillance system (section 3.3.2). For clinical laboratories that
require additional training, the NRL could conduct training on site.
Ideally, the NRL should organize annual meetings of representatives of clinical laboratories
to discuss the performance in the network during the year, new features and methods
and to update information on AMR. If necessary, a practical session could be included,
especially for new clinical laboratories.
In countries in which the NRL is still building its capacity to support the surveillance
system, support from and collaboration with the WHO AMR Surveillance and Quality
Assessment Collaborating Centres Network (4) could be useful. This could include
twinning agreements between countries and NRLs to develop capacity, methods,
technical activities and surveillance capacity.

15
GLASS guidance for national reference laboratories

06 Data collection and analysis

6.1 Laboratory The NRL should have a system in which surveillance sites register information on patients
information systems and specimens, the tests ordered, test results and reports. The database should store
and test reports unique patient and specimen identifiers and requests for testing of submitted isolates
or specimens. WHONET is a useful tool for countries with no laboratory information
system at the early stages of a national surveillance system. It should be backed up
and made secure to ensure patient privacy. Surveillance sites should share information
with the NRL in the national surveillance system, so that the quality of the data can be
verified and the data analysed at national level.
NRLs should use the laboratory information system or another program (e.g. WHONET)
to collect data on isolates referred for confirmation and any additional analyses, and
should report all test results to the submitting laboratory or institution. Electronic
reporting of results allows the collection, collation and analysis of data to provide timely
information for local infection prevention and to ensure that the results from submitting
institutions are complete. It also provides an incentive for continued submission of
specimens and isolates from laboratories. WHONET software is freely available and is
suitable for the surveillance and analysis of AMR required by GLASS.

16
 06 Data collection and analysis

6.2 Data analysis The clinical laboratories that support surveillance sites should (i) record receipt of
for the national specimens upon arrival; (ii) ensure processing of specimens according to standard
surveillance of operating procedures; (iii) read and record results, including interpretation and confirmation
antimicrobial by laboratory staff; (iv) provide clinicians with timely results and advice on patient
management; (v) provide on-call services for follow-up requests, queries from clinical
resistance
teams and urgent requests for testing outside working hours; (vi) provide accurate, timely
data to surveillance staff; and (vii) implement and enforce quality control procedures.
Clinical laboratories have an important role in diagnostic stewardship (14), and the NRL
has a key role in supporting and ensuring that clinical laboratories develop such capacity.
At surveillance sites, staff of the clinical, microbiology laboratory and for surveillance
and epidemiology should collaborate in reporting data to the national AMR surveillance
coordinating centre. Furthermore, the NRL and the national coordinating centre should
work in tandem with surveillance sites to ensure the quality and correct interpretation
of AMR data (Fig. 3) (14). NRLs should work with the national coordinating centre to
assist in analysis of national data on AMR, for which WHONET could be used.

Fig. 3. Relations between individual care and surveillance data

Patient’s specimen
Laboratory
Clinical team
team
Patient’s laboratory results

Patient’s
laboratory
results
Empirical Local AMR
Surveillance
treatment control
guidelines strategies
team

National AMR
Surveillance data surveillance
system

17
GLASS guidance for national reference laboratories

07 Assessment
of laboratories

The NRL and the national coordinating centre should establish a programme to monitor
the performance and activities of surveillance sites that includes all stakeholders in the
system, such as laboratory staff, clinicians and epidemiologists. The NRL should use a
standard method to monitor surveillance sites, such as the GLASS laboratory assessment
questionnaire for AMR testing (11), to assess and evaluate the activities, QC and performance
of laboratories. An assessment visit indicates areas that require support, such as additional
training, supplies or data analysis. The results of the assessment should be used to analyse
the entire laboratory network, identify where improvements could be made and plan activities
to improve the surveillance system.

18
 GLASS guidance for national reference laboratories

08
References

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National antimicrobial resistance surveillance systems and participation in the Global
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