Report Who Eqas 2009-Final

The External Quality Assurance System of the
WHO Global Foodborne Infections Network 2009

WHO Collaborating Centre for Antimicrobial Resistance in Foodborne Pathogens
National Food Institute

Kemitorvet, Kgs. Lyngby, Denmark
Rene Hendriksen, Susanne Karlsmose, Valeria Bortolaia, Arne Bent Jensen, Frank Aarestrup
THE EXTERNAL QUALITY ASSURANCE SYSTEM OF

THE WHO GLOBAL FOODBORNE INFECTIONS NETWORK
YEAR 2009
DATE OF ISSUE: DECEMBER 2010
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THE EXTERNAL QUALITY ASSURANCE SYSTEM OF THE WHO GLOBAL
FOODBORNE INFECTIONS NETWORK YEAR 2009
1. edition, December 2010

Copyright: National Food Institute, Technical University of Denmark
Photo: Mikkel Adsbøl
ISBN: 978-87-92158-88-8
The report is available at

www.food.dtu.dk

Technical University of Denmark
Kemitorvet
Building 204
2800 Kgs. Lyngby
Denmark
Tel: +45 35 88 70 00
Fax +45 35 88 70 01
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Contents
List of Abbreviations ..................................................................................................................................... 1

1. Introduction ................................................................................................................................................ 2
2. Materials and Methods ............................................................................................................................... 3
2.1 Participants ........................................................................................................................................... 3
2.2 Strains ................................................................................................................................................... 3
2.3 Antimicrobials ...................................................................................................................................... 4
2.4 Distribution ........................................................................................................................................... 4
2.5 Procedure .............................................................................................................................................. 5
3. Results ........................................................................................................................................................ 6
3.1 Methods used by EQAS participants .................................................................................................... 7
3.2 Serogrouping and serotyping of Salmonella strains ............................................................................. 7
3.3 Antimicrobial susceptibility testing (AST) of Salmonella strains ........................................................ 9
3.4 Serogrouping and serotyping of Shigella strains ................................................................................ 10
3.5 Antimicrobial susceptibility testing (AST) of Shigella strains........................................................... 10
3.6 Identification of Campylobacter strains ............................................................................................. 11
3.7 MIC determination of Campylobacter strains .................................................................................... 12
3.8 Identification of the unknown culture ................................................................................................ 12
4. Discussion ................................................................................................................................................ 13
4.1 Serogrouping and serotyping of Salmonella strains ........................................................................... 13
4.2 Antimicrobial susceptibility testing (AST) of Salmonella strains ...................................................... 14
4.3 Serogrouping and serotyping of Shigella strains ................................................................................ 17
4.4 Antimicrobial susceptibility testing (AST) of Shigella strains........................................................... 17
4.5 Identification of Campylobacter strains ............................................................................................. 18
4.6 Antimicrobial susceptibility testing (AST) of Campylobacter strains ............................................... 18
4.7 Identification of the unknown culture ................................................................................................ 19
5. Conclusions .............................................................................................................................................. 19
Reference List .............................................................................................................................................. 21
Figure and Tables ......................................................................................................................................... 22
Appendixes (1, 2, 3, 4a, 4b) ......................................................................................................................... 45
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List of Abbreviations
AMC, Amoxicillin / Clavulanic acid
AMP, Ampicillin
AST, Antimicrobial Susceptibility Testing
CAZ, Ceftazidime
CCM, Czech Collection of Micro-organisms
CHL, Chloramphenicol
CIP, Ciprofloxacin
POD, Cefpodoxime
CRO, Ceftriaxone
CTX, Cefotaxime
DTU Food, Danish Technical University - National Food Institute
ENR, Enrofloxacin
EQAS, External Quality Assurance System
ERY, Erythromycin
EUCAST, The European Committee on Antimicrobial Susceptibility Testing
FFN, Florfenicol
FIS, Sulfisoxazole
GEN, Gentamicin
KAN, Kanamycin
MIC, Minimum Inhibitory Concentration
NAL, Nalidixic Acid
QC, Quality Control
SMX, Sulfametoxazole
STR, Streptomycin
SXT, Trimethoprim + Sulphonamides
TET, Tetracycline
TMP, Trimethoprim
WHO, World Health Organization
WHO GFN, WHO Global Foodborne Infections Network
WHO GFN CDB, WHO GFN Country Databank
WHO GSS, WHO Global Salm-Surv
XNL, Ceftiofur
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1. Introduction
In J anuary 2000, W HO l aunched the WHO Gl obal S alm-Surv ( WHO-GSS) which represents an
international e ffort aiming to e nhance l aboratory-based surveillance of Salmonella infections a nd to
promote p revention and c ontrol a ctivities. This Program, which ha s b een renamed “the W HO Global
Foodborne Infections N etwork ( WHO G FN)”, focuses on enhancing WHO M ember S tates’ capacity to
detect a nd r espond t o f oodborne disease outbreaks b y c onducting l aboratory-based surveillance of
Salmonella and other foodborne pathogens. Since its inception, the scope of WHO GFN has expanded to
include additional foodborne pathogens like Shigella and Campylobacter. Salmonella, Campylobacter and
Shigella are among the most important foodborne pathogens worldwide and account for millions of cases
of diarrheal disease a nd t housands of d eaths pe r year, i mpacting bot h developing a nd i ndustrialized
countries. Furthermore, the increased num ber of Salmonella and Shigella isolates which are r esistant to
antimicrobials i s of m ajor conc ern since t hese i solates are associated with infections cha racterized by
increased morbidity and mortality.
An External Quality Assurance System (EQAS) program was established in 2000 to support participation
of l aboratories in the W HO GFN. The original goal of t his pr ogram w as t o a ssess t he qua lity of
Salmonella serotyping a nd antimicrobial s usceptibility te sting ( AST) data pr oduced by M ember States
and t o enhance t he r eliability of these data b y i dentifying areas w hich c ould be nefit f rom a dditional
support. In 2003, t he EQAS program w as e xpanded t o i nclude additional foodborne pa thogens, as
mentioned above. The number of participants submitting data related to one or more components of the
EQAS i ncreased f rom 44 l aboratories i n 2000 to 180 l aboratories i n 20 09. According t o a goal s et b y
WHO GFN, all national reference laboratories should perform Salmonella serotyping with a maximum of
one deviation out of eight strains tested (error rate of 13%) and AST with a maximum error rate of 10%
(either <5% very major / major errors and <5% minor errors, or <10% minor errors, as defined further in
this report).
The EQAS is organized annually by the National Food Institute (DTU Food), Kgs. Lyngby, Denmark in
collaboration w ith C enters f or D isease C ontrol a nd P revention ( CDC) i n A tlanta, U SA; W orld H ealth
Organization ( WHO) i n G eneva, S witzerland; P ublic H ealth A gency of C anada (PHAC) in C anada;
National Salmonella and Shigella Center (NSSC), National Institute of Health, Department of Medical
Sciences i n Thailand and Institute P asteur ( IP) i n Paris, France. The t echnical a dvisory group f or t he
WHO EQAS program consists of members of the WHO GFN Steering Committee.
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Individual l aboratory da ta are confidential a nd only known by t he participating l aboratory, t he EQAS
Organizer (DTU Food) and the respective WHO GFN regional centre. All summary conclusions are made
public.
2. Materials and Methods

2.1 Participants
A pr e-notification a nnouncement of t he EQAS 2009 was m ade t hrough t he W HO G FN l ist s erver on
April 21, 2009 a nd a reminder was sent on May 4, 2009 ( App. 1). The pre-notification was available in
English, Spanish, Portuguese, French, Chinese and Russian, and included invitations to participate in the
EQAS 2009 program f or s erotyping a nd AST of Salmonella and Shigella, i dentification a nd AST
[Minimum I nhibitory C oncentration (MIC) determination] of Campylobacter, a nd i dentification of a n
unknown foodborne pathogen. Participation was free of charge, but each laboratory was expected to cover
expenses associated with the analyses performed.
2.2 Strains
Eight Salmonella strains, f our Shigella strains, and t wo Campylobacter strains were s elected for t he
EQAS 2009 from the DTU Food’s strain collection. The unknown foodborne pathogen, a Vibrio mimicus
strain, was s elected by the Laboratory subcommittee unde r the W HO G FN S teering C ommittee, and it
was provided by the US-CDC. Individual sets of Salmonella and Shigella strains were inoculated as agar
stab c ultures i n nut rient a gar, while the Vibrio mimicus strain was i noculated as a gar s tab cul tures in
Tryptic S oy Agar. T he Campylobacter strains were l yophilized in glass vi als by Czech Collection of
Micro-organisms (CCM), Czech Republic. The serotype of each Salmonella strain was designated on the
basis of O (somatic) and phase 1 and phase 2 H (flagellar) antigens according to the scheme of Kaufmann-
White (2007) (5). The Salmonella serotype was determined by DTU Food and verified by the CDC and IP
prior to distribution. The antimicrobial susceptibility pattern of the Salmonella strains was determined by
DTU F ood and ve rified by C DC. T he Shigella serotype w as pe rformed by P HAC and verified by the
NCCS. DTU Food determined the antimicrobial susceptibility pattern of the Shigella strains, which was
verified by PHAC and CDC. Finally, all results were later confirmed at DTU Food (apart from Shigella
serotyping which is not routinely performed at DTU Food).
Furthermore, laboratories which did not formerly participate in WHO GFN EQAS AST component were
provided with lyophilized international reference strains, namely E. coli CCM 3954 ~ ATCC 25922 and
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C. jejuni CCM 6214 ~ ATCC 33560, which were purchased at the Czech Collection of Micro-organisms
(CCM); The Czech Republic.
2.3 Antimicrobials
AST of the Salmonella, Shigella, and Campylobacter strains was performed a t t he D TU F ood, and t he
obtained results were used as a reference standard (App. 2). The following antimicrobials were used in the
EQAS 2009 for AST of Salmonella and Shigella strains: ampicillin, AMP; cefotaxime, CTX; ceftazidime,
CAZ; ceftriaxone, C RO; c hloramphenicol, C HL; c iprofloxacin, C IP; gentamicin, GEN; n alidixic a cid,
NAL; s treptomycin, STR; sulfamethoxazole, SMX; te tracycline, TET; tr imethoprim, TMP a nd
trimethoprim + s ulphonamides, S XT. In a ddition, i t w as pos sible t o c onfirm t he pr esence o f ESBL-
producing strains by using the antimicrobials CTX and CAZ in combination with the inhibitor clavulanic
acid. T he f ollowing a ntimicrobials w ere us ed i n t he EQAS 2009 for AST of Campylobacter strains:
chloramphenicol, CHL; ciprofloxacin, CIP; erythromycin, ERY; gentamicin, GEN; nalidixic acid, NAL;
streptomycin, STR and tetracycline, TET.
MIC determination was performed by using Sensititre systems from Trek diagnostics Ltd, and guidelines
and br eakpoints by Clinical a nd Laboratory S tandards Institute ( CLSI) based on doc ument M07-A7
(2006) “Methods fo r Dilution Antimicrobial S usceptibility T ests f or B acteria T hat G row A erobically”;
Approved S tandard - Seventh E dition (4), doc ument M 100-S19 ( 2009) “Performance S tandards f or
Antimicrobial S usceptibility T esting”; N ineteenth Informational S upplement (3), document M 31-A3
(2008) “ Performance S tandards f or A ntimicrobial D isk and Dilution Susceptibility T ests f or Bacterial
Isolated from Animals”; Approved Standard - Third Edition (2), and document M45-A (2006) “Methods
for A ntimicrobial D ilution and Disk Susceptibility Testing of Infrequently Isolated or F astidious
Bacteria”; Approved Standard – First Edition (1). Guideline were used for interpretation of AST results
with the exception of i) cefotaxime, ceftazidime and ci profloxacin susceptibility te sting for w hich
EUCAST (www.eucast.org) epidemiological cut-off values were utilized; ii) streptomycin and ceftriaxone
susceptibility te sting for w hich DTU Food i nterpretative cr iteria w ere utilized; a nd iii) Campylobacter
AST, for w hich EUCAST e pidemiological cut-off va lues were us ed. A ll br eakpoints a re l isted i n t he
protocol (App. 3).
2.4 Distribution
Bacterial cultures were e nclosed i n doubl e pa ck c ontainers ( class U N 6,2) a nd s ent t o pa rticipating
laboratories a ccording t o t he International A ir T ransport A ssociation (IATA) r egulations a s “Biological
Substance c ategory B” classified U N3373. P rior t o s hipping, laboratories w ere i nformed about t he
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dispatch da te. Import pe rmits w ere ne cessary f or s hipping t he pa rcels t o a l arge num ber of countries.
Many of the parcels were shipped as “overpack” through international hubs which offered to support the
costs of f urther di stributing t he p arcels. H elen Tabor f rom P HAC; C anada, M att M ikoleit f rom CDC;
United S tates, A roon Bangtrakulnonth from N SSC; T hailand, E nrique P erez f rom H ealth S urveillance,
Disease P revention and Control; Brazil, Francois Xavier W eill from IP; France, Rita Tolli from Istituto
Zooprofilattico S perimentale de lle R egioni Lazio e T oscana, Italy and Changwen K e f rom C enter f or
Disease Control and Prevention of Guangdong Province, China shipped to all Canadian, North American,
Thai, Latin American, Francophone African, Italian and Chinese institutes, respectively. The first parcel
was dispatched from DTU Food on August 25, 2009 and the last on November 12, 2009.
2.5 Procedure
Participants were i nstructed t o dow nload t he pr otocol a nd a dditional doc uments ( App. 4a a nd 4b ;
available only in English) from http://www.antimicrobialresistance.dk/. In addition, they were requested
to s ubculture t he s trains pr ior t o performing the method routinely us ed in their l aboratory. T he EQAS
2009 components included s erotyping and AST of e ight Salmonella and f our Shigella strains,
identification a nd MIC de termination of t wo Campylobacter strains, AST of t wo qua lity c ontrol (QC)
strains ( E. coli CCM3954 / A TCC25922, C. jejuni CCM 6214 / A TCC33560), a nd i dentification of a n
unknown foodborne pathogen (Vibrio mimicus). Furthermore, the laboratories were requested to save and
maintain the ATCC reference strains for future proficiency tests (App. 4a and 4b).
After performing the te sts, participants were requested to enter t he obt ained results (serotype and / or
serogroup, MIC values or zone-diameter in millimeters, and antimicrobial susceptibility categories of the
Salmonella and Shigella strains; identification, MIC values, and antimicrobial susceptibility categories of
the Campylobacter strains; and identification of the unknown sample) into an electronic record sheet in
the WHO GFN web-based database through a secured individual login, or alternatively, to send the record
sheets from the enclosed protocol by fax to DTU Food. The database was activated on September 7, 2009
and closed on March 17, 2010.
The Salmonella, Shigella and Campylobacter strains were categorized as resistant (R), intermediate (I) or
susceptible (S) to all tested antimicrobials. The interpretative criteria followed to generate the results used
as reference standard were based on both clinical breakpoints and epidemiological cut-off values.
Of note, the terms ‘susceptible’, ‘intermediate’ and ‘resistant’ should be reserved for classifications made
in r elation t o t he t herapeutic a pplication of antimicrobial a gents. When r eporting d ata b ased on
epidemiological cut-off values, bacteria should instead be reported as ‘wild-type’ or ‘non-wild-type’ (7).
Due t o t he di fferent AST methods us ed b y t he participants and to simplify interpretation of the results,
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throughout thi s r eport w e w ill ma intain the te rms s usceptible, intermediate and resistant also when we
refer to wild-type and non-wild-type strains.
Susceptibility results had to be interpreted on a n individual basis for each antimicrobial tested, with the
exception of cephalosporins which were interpreted according to CLSI Approved Standard – Nineteenth
Edition, doc ument M 100-S19 ( 2009) “Performance S tandards f or A ntimicrobial S usceptibility T esting,
Table 2A ”. Participants were i nstructed to use t he Salmonella / Shigella antisera and the antimicrobials
used i n the methods routinely p erformed. In a ddition, t hey w ere i nstructed t o us e t heir usual standard
breakpoints f or c ategorizing t he results obtained by A ST. All l aboratories w ere r equested to enter M IC
values for the C. jejuni (ATCC 33560) reference strain, and either zone diameters or M IC values for the
E. coli (ATCC 25922) reference s train. A fter s ubmitting t he results, participants were i nstructed t o
retrieve an instantly generated report from the secure web site. This report was created on an individual
basis, and reported all deviations f rom t he expected results and suggestions for s olving or investigating
the cause o f er ror. D eviations of antimicrobial s usceptibility test results from t he ex pected results were
categorized a s m inor, major or ve ry m ajor. Minor de viations a re d efined as classification of an
intermediate strain as susceptible, resistant or vi ce v ersa ( i.e. I ↔ S o r I ↔R). Major d eviation is the
classification of a susceptible strain as resistant (i.e. S → R). Very major deviation is the classification of
a resistant strain as susceptible (i.e. R → S). In this report, the deviations of AST results are divided into
two categories, i.e. critical deviations which include major and very major deviations, and total deviations
which include also the minor deviations.
3. Results
A t otal of 192 l aboratories responded t o t he pr e-notification and were enrolled i n t he EQAS. W hen t he
deadline f or s ubmitting results w as r eached, 18 0 l aboratories i n 90 c ountries ha d upl oaded da ta. T he
following countries provided data for at least one of the EQAS components (Figure 1): Albania, Algeria,
Argentina, A ustralia, Barbados, Belarus, Belgium, B olivia, Bosnia a nd H erzegovina, Brazil, B runei
Darussalam, Bulgaria, Cambodia, Cameroon, Canada, Central African Republic, Chile, China, Colombia,
Democratic Republic of Congo, Costa Rica, Croatia, Cuba, Cyprus, Czech Republic, Denmark, Ecuador,
Egypt, E stonia, E thiopia, F inland, F rance, G ambia, G eorgia, G ermany, Greece, G uatemala, H onduras,
Hungary, India, Iran, Ireland, Israel, Italy, Ivory Coast, Jamaica, Japan, Jordan, Kenya, Korea, Lao PDR,
Lithuania, Luxembourg, Madagascar, Malaysia, Malta, Mauritius, Mexico, Moldova, Morocco, Namibia,
New Zealand, N icaragua, N igeria, S ultanate of O man, P anama, P araguay, P eru, P hilippines, Poland,
Russia, Serbia, S ingapore, S lovakia, S lovenia, South A frica, S ri Lanka, S udan, S uriname, T aiwan,
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Thailand, Trinidad and Tobago, Tunisia, Turkey, United Kingdom, Uruguay, USA, Venezuela, Vietnam,
Yemen.
In the description of results, arbitrary thresholds of quality limits were not used. The results for AST are
expressed as correct, minor, major, very major, and critical and total deviations as described above.
3.1 Methods used by EQAS participants

Of a tot al of 182 laboratories receiving Salmonella strains, 161 ( 88%) pa rticipated i n t he Salmonella
serogrouping component of t he EQAS, and 153 (95%) pa rticipated i n t he complete s erotype m odule of
the EQAS. In addition, 153 (84%) laboratories submitted AST results. Among the laboratories performing
AST, 129 (84%) submitted results for the quality control (QC) strain E. coli ATCC 25922. T he majority
(n=102; 79%) of t hese l aboratories us ed t he di sk di ffusion m ethod, w hile a MIC determination method
was utilized by a smaller number (n=27; 21%) of laboratories.
Of 136 l aboratories r eceiving Shigella strains, 118 (87%) s ubmitted Shigella serogroup r esults
(speciation) and 82 (69%) of these laboratories serogrouping t he i solates further analyzed t he strains to
the serotype level. In addition, Shigella AST was performed by 111 (82%) laboratories.
All pa rticipating la boratories w ere given information r egarding the MIC br eakpoints us ed f or
interpretation when generating the expected values, with the exception of equivalent breakpoints for disk
diffusion. I n a ddition, all participating laboratories were i nstructed on interpreting resistance t o third
generation cephalosporins and to fluoroquinolones.
Of the 131 l aboratories receiving Campylobacter strains, 87 (66%) reported identification results and 25
(19%) submitted AST results for both Campylobacter strains.
Of the 146 laboratories receiving the unknown culture for identification, 56 (38%) submitted results.
3.2 Serogrouping and serotyping of Salmonella strains

In 2009, the percentage of laboratories reporting complete serotype results for all eight strains increased to
83% (n=119), thus contrasting with the decreasing trend observed in the two previous years (e.g. in 2008:
66%, n=100). The proportion of correctly serotyped strains increased from 83% (n=888) in 2008 to 86%
(n=974) in 2009 (Table 1).
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In Table 2 , the num ber of pa rticipating l aboratories is reported according t o the num ber o f co rrectly
serotyped samples. In 2009, 76 (50%) of the 153 participating la boratories s erotyped all e ight s trains
correctly, and 29 (19%) laboratories correctly serotyped seven of the eight strains. Summarizing, in 2009,
a total of 105 (69%) participating laboratories met the threshold for adequate performance of Salmonella
serotyping, which r epresents a considerable increase com pared t o 2008 when only 86 ( 57%) of t he
participating la boratories met the performance quality t hreshold. In addition, 82% of the pa rticipating
laboratories c orrectly ide ntified half of the s trains, which represents an 8% increase c ompared t o 2008
(74%).
In Table 3 , t he pe rformance of Salmonella serotyping is r eported on a region-based categorization of

participating laboratories. Overall, the a ccuracy of s erotyping increased in m any regions compared to
2008. The African, Latin American and Southeast Asian regions experienced the largest influx of EQAS
participants w ith up t o five ne w pa rticipants c ompared t o 2008. I n c ontrast, in the European r egion,
participation to the EQAS 2009 decreased of three laboratories compared to 2008.
The num ber of tested strains increased mostly in the A frican, Latin American, and S outheast Asian
regions and in China which tested between 18 and 55 additional strains in 2009. T he a ccuracy of
serotyping i ncreased m ostly i n laboratories f rom A frica, Caribbean, E urope, and Latin American
(between 4.7% and 26.2% increase c ompared to 2008) . A de crease i n accuracy of s erotyping was
observed in North America and the Asia & Middle Eastern region, with the most considerable decrease in
Asia & Middle Eastern region (14.9% decrease compared with 2008).
The ove rall pe rformance of laboratories performing Salmonella serogrouping was satisfactory, as t he
percentages of deviations were very low for almost all tested strains, ranging from 0.6% (WHO S9.8) to
10.9% ( WHO S 9.6). By excluding s train WHO S 9.6, t he range o f de viations was from 0.6% t o 3.8 %
(Table 4).
Of 151 laboratories performing s erotyping of the int ernal qua lity control s train (WHO S 9.1, used in
EQAS 2000, 2001, 2004 , 2006, 2007 and 2008), 141 (93.4%) reported a correct result, thus leading to a
deviation r ate of onl y 6. 6% (Table 4 ). The ability of pa rticipating la boratories to correctly serotype t he
internal qua lity control strain was consistent through di fferent years, and r anged from 95% i n 20 04 t o
93% in 2009 even though the number of participating laboratories increased (Table 5).
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Deviations in Salmonella serotyping ranged from 6.6% (WHO S9.1) to 19% (WHO S9.2) (Table 4), thus
showing an i mprovement c ompared t o last y ear when the hi ghest pe rcentage of de viations w as 3 4.1%,
and deviations greater than 20% were reported for four strains. In 2009, the three strains resulting in most
deviations w ere W HO S9.2: Salmonella Brandenburg ( I 4,12: l,v:e,n,z15) w ith 19 % deviations, WHO
S9.6: Salmonella Worthington ( I 1,13,23:z:l,w) w ith 16.3 % de viations, a nd W HO S 9.4: Salmonella
Bredeney (I 1,4,12:l,v:1,7) with 16% deviations (Table 4). Only WHO S9.1: Salmonella Enteritidis strain
was serotyped satisfactorily with 6.6% deviations which is a slight decrease compared to 2008 (Table 4).
3.3 Antimicrobial susceptibility testing (AST) of Salmonella strains

A tot al of 12,707 antimicrobial s usceptibility te sts was performed in 2009 b y 153 pa rticipating
laboratories. Of the submitted results, 94% w ere in agreement w ith the e xpected result, which i s a 3%
improvement compared to 2008 (Table 6). Minor, major and very major deviations were observed in 3%,
2% and 1% of the submitted results, respectively (Table 6).
No major di fficulties in a ssessing antimicrobial s usceptibility w ere e ncountered for a ny of the te sted
combinations of strains and antimicrobials. Mostly, difficulties w ere ex perienced in assessing
susceptibility to STR, SMX and TET (Table 7).
Major deviations categorized by tested antimicrobial are reported in Table 8. Notably, a large number of
critical deviations was observed for CIP (8%), STR (9%) and SMX (7%). These antimicrobials together
with TET resulted also in very high numbers of total deviations (Table 8). In 2009, the average number of
critical a nd total de viations overall observed was 3% an d 6%, respectively, which represents an
improvement compared to 2008.
The 2009 E QAS trial did not include any ESBL-producing Salmonella strain. However, participants had
1% and 2% deviating results for CAZ and CRO, and for CTX, respectively (Table 8).
In 2009, t he num ber of laboratory participating t o the AST component of E QAS decreased in Central
Asia & Middle East, Caribbean, China, Europe, and North America (Table 9). In particular, compared to
2008, t he Europe and the C entral A sia & M iddle E ast r egions registered a decrease of 11 a nd 6
participants, respectively. By contrast, additional eight l aboratories took pa rt t o t he EQAS AST
component in the Southeast Asian region. Overall, the performance of AST improved in all regions, most
notably i n t he A frican a nd t he C entral A sia & M iddle E astern r egions. Overall, antimicrobial
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susceptibility te st r esults were r eported correctly in percentages r anging from 90.1 % (Africa) t o 98.7%
(North America) (Table 9).
Antimicrobial s usceptibility of E. coli ATCC 25922 w as t ested b y 2 7 l aboratories with the MIC
determination method a nd b y 102 laboratories with t he di sk di ffusion m ethod. The pr oportion of
laboratories which submitted values outside the acceptable interval for the reference strain E. coli ATCC
25922 i s r eported i n T able 10. The pe rcentages of l aboratories which reported MIC values out side the
intervals acc epted for t he Q C s train ranged from 5% to 21% for S TR and F IS susceptibility testing,
respectively (Table 10) . In g eneral, laboratories using t he M IC de termination m ethod reported va lues
within the acceptable interval in higher percentages compared to the laboratories using the disk diffusion
method, with the exception of CTX, CIP, and FIS susceptibility testing (Table 10).
3.4 Serogrouping and serotyping of Shigella strains

In 2009, t he pe rformance of Shigella speciation was satisfactory, as t he pe rcentages of de viations were
very l ow f or a ll t he f our t est s trains, r anging f rom 0.9% ( WHO S H 9.1 - 9.3) t o 4.6% ( WHO S H 9.4)
(Table 11 ). Similar r esults w ere obs erved among l aboratories t hat p erformed f ull s erotyping. Thus, t he
percentages of deviations in Shigella serotyping ranged from 4.2% (WHO SH 9.3) to 12.5% (WHO SH
9.4), w hich r epresents an i mprovement c ompared t o t he pi lot s tudy performed in 2008. T he s train
resulting in most deviations was WHO SH 9.4: Shigella boydii serotype 2, which was reported as serotype
1 by eight participating laboratories.
In Table 12, the performance of Shigella serotyping is reported according to geographical distribution of
participating laboratories. The majority of participating laboratories was located in Latin America (n=16),
Europe ( n= 15), C hina ( n= 13) and S outheast A sia ( n= 11). The a ccuracy of Shigella serotyping r esults
ranged from 72.2% (Africa) to 100% (Oceanic, China, Central Asia & Middle East).
3.5 Antimicrobial susceptibility testing (AST) of Shigella strains

A t otal of 4,548 a ntimicrobial s usceptibility t ests w ere pe rformed i n 2009 b y 111 pa rticipating
laboratories. Agreement with the expected result was achieved in 96% of the reported results, which is a
1% improvement compared to 2008 (Table 13). Minor, major and very major deviations were observed in
2%, 1% and 1% of reported results, respectively (Table 13).
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No major di fficulties in a ssessing antimicrobial s usceptibility w ere en countered for an y of t he t ested
combinations of strains and antimicrobials (Table 14). STR and TET accounted for 18.1% and 7. 5% of
total deviations, respectively (Table 15).
ESBL-producing Shigella strains were not i ncluded i n t he EQAS 2009 trial. However, t he participating
laboratories had between 0.3% and 1.1% deviating results for CAZ, CRO, and CTX (Table 15).
In 2009, l aboratories i n almost a ll r egions pa rticipated i n t he Shigella AST component, except f or the
Oceanic region. The majority of participating laboratories was located in the European, Latin American,
Southeast A sian and African regions where 22, 20, 18 a nd 17 laboratories pa rticipated to this E QAS
iteration, respectively ( Table 1 6). By c onsidering participating laboratories in relation to their
geographical location, the percentage of correct AST results ranged from 93.3% (Africa) to 100% (North
America). The African and Central A sia & M iddle E ast regions reported results pr esenting t he hi ghest
percentages of critical a nd t otal de viations, i.e. 4.3% a nd 4.4% c ritical deviations, and 6.8% a nd 5.2%
total de viations, r espectively. Also the S outheast A sian r egion h ad a considerably high number o f t otal
deviations (5.9%) (Table 16).
3.6 Identification of Campylobacter strains

Participation in the EQAS 2009 Campylobacter component was requested by 131 l aboratories, but only
86 (66%) submitted results within the deadline. Of the participating laboratories, 77% and 95% performed
correct species identification for strain #1 (C. coli) and #2 (C. jejuni), respectively (Table 17). This is the
first time in the EQAS program that a Campylobacter strain has been correctly identified by such a high
percentage ( 95%) of laboratories. Only f our d eviations w ere reported, namely three C. upsaliensis and
one C. lari.
In Table 18, the p erformance of Campylobacter identification i s r eported a ccording t o geographical

location of pa rticipating laboratories. The ma jority (n= 28; 33 %) of participating laboratories w ere in
Europe ( n= 28), but participation from C hina a nd Latin America [12 (14%) and 14 (16%) laboratories,
respectively] was al so considerable. The a ccuracy in Campylobacter identification ranged f rom 40%
(Central A sia & M iddle East) t o 100% ( Oceanic, R ussia, and Caribbean). A high num ber of deviations
was observed in the African region where only 53.8% of the strains were correctly identified.
11
3.7 MIC determination of Campylobacter strains
A t otal of 292 M IC determinations was performed i n 2009 by 25 participating laboratories. Among t he
reported r esults, 91.4% w ere i n a greement w ith the ex pected result ( Table 19). Major and ve ry m ajor
deviations were observed in 4.5% and 4.1% of reported results (Table 19).
No major di fficulties in a ssessing antimicrobial s usceptibility w ere e ncountered for an y of t he t ested

combinations of strains a nd antimicrobials (Table 20) . However, 11.8%, 11.1%, 10.8% a nd 9.8%,
deviations were reported for STR, TET, ERY and NAL susceptibility testing, respectively (Table 21).
In 2009, MIC va lues w ere submitted by la boratories in Africa, C hina, E urope, N orth A merica, Latin
American, and Southeast Asia (Table 22). Agreement with expected values was observed in percentages
ranging from 42.9% (Africa) t o 100% (North America) (Table 22). The highest percentages of critical
deviations were reported from laboratories in the African and Southeast Asian regions (50% and 28.6%,
respectively; Table 22).
MIC values of reference strain C. jejuni ATCC 33560 were tested by 24 (96%) laboratories. Of these, 15
laboratories used micro-dilution procedures, while 9 laboratories used agar-dilution procedures and tested
only C IP, E RY and GEN. Overall, the percentage of l aboratories w hich s ubmitted va lues within the
acceptable interval for the reference strain ranged from 72.7% to 91.7% (for ERY and NAL susceptibility
testing, respectively; T able 23) . Of not e, only 40% of t he l aboratories us ing a gar-dilution w ith a n
incubation temperature of 42°C met the quality control interval for ERY susceptibility testing (Table 23).
3.8 Identification of the unknown culture

Identification of the un known e nteric p athogen ( Vibrio mimicus) was performed b y 5 6 laboratories.
Overall, 75% of t he pa rticipating l aboratories identified the s train as a Vibrio spp. Only 27 ( 48%)
laboratories completely and correctly identified the strain. Thirteen (23%) laboratories reported deviating
results, namely Campylobacter spp. (n=2), Arizona spp. (n=1) Staphylococcus spp. (n=1) Aeromonas
hydrophila (n=1), Salmonella enterica Onderstepoort (n=1), Aeromonas salmonicida salmonicida (n=1),
Enterobacter hafnia alvei (n=1), Enterobacter cloacae (n=1), Micrococcus luteus (n=1), Escherichia coli
(n=1), and Bacillus spp. (n=1).
12
4. Discussion
4.1 Serogrouping and serotyping of Salmonella strains
As i n pr evious years, the s election of s erovars included i n the 2009 WHO GF N E QAS t rial was ba sed
both on t he 15 m ost common s erovars s ubmitted t o t he WHO G FN C ountry D ata Base ( CDB)
http://www.antimicrobialresistance.dk and on various reports and scientific publications. To facilitate the
global assessment of Salmonella serotyping capacity, we chose serovars which may be very common in
certain regions and sporadically encountered i n other regions. In 2009, we i ncluded Salmonella serovar
Brandenburg w hich i s r anked a mong t he t op 15 m ost c ommon s erovars i n the O ceanic r egion, more
specifically in New Zealand where it has been reported as a frequently isolated serovar among humans for
more than a decade (6). S. Brandenburg has also been often observed in Europe. In addition, we included
Salmonella Bredeney which is frequently reported in Europe, and Salmonella Muenster which is reported
occasionally in Europe and the United States but is very common in Africa. Then, we included
Salmonella serovars S andiego and W orthington which a re c ommon i n t he Latin A merican region. We
also included S. Stanley which could be defined endemic in Southeast Asia, and it was the second most
common serovar between 2002 and 2007 in Thailand. Notably, many of the S. Stanley observed in Europe
are isolated from human patients travelling to the Southeast Asian region. Finally, we included S. Albany
which is associated with the Southeast Asian region too.
The number o f l aboratories w hich correctly serotyped all e ight Salmonella strains i ncreased from 100
(66%) in 2008 t o 119 (83%) in 2009, which represents the second best performance after the first EQAS
in 2000 (Table 1) . Similarly, t he pe rcentage of c orrectly s erotyped strains was higher only in 20 07 and
2002 when, however, fewer laboratories submitted results compared to 2009. Two reasons could explain
the excellent results obtained in 2009. First, all the Salmonella strains selected for the EQAS 2009 could
be fully s erotyped using c ommonly a vailable a ntisera. Second, the i ncreased number o f WHO GF N
capacity bui lding l aboratory t raining c ourses i n both A frica a nd Latin A merica may have pr ovided a
better understanding of s erotyping methods and improved knowledge of t he availability of good q uality
antisera.
Of not e, onl y 93% of participating laboratories correctly s erotyped the internal cont rol s train (WHO
S9.1), which is the lowest percentage obtained since 2001 (Table 5). This apparent contradiction with the
otherwise excellent general performance support the hypothesis that the strains provided in 2009 were of
easier ide ntification compared to the te st s trains of previous years. T he qua lity threshold of correctly
serotyping at least seven strains was met by 69% of participating laboratories, thus demonstrating a clear
13
improvement c ompared to 2008. Once m ore, this r esult emphasizes t hat t he pa nel o f strains chosen in
2009 was easier to serotype compared to the strains provided in 2008.
In general, the obtained results indicate that most laboratories worldwide have the capacity to serotype the
most c ommon Salmonella serovars. However, t he data al so show that one r egion, i.e. Central A sia &
Middle E ast, still lacks access t o reliable ant isera and laboratory t raining cou rses ne cessary t o identify
regionally prevalent serovars, as this region reported the highest percentages of deviations of serotyping
results. Noteworthy, m any regions obtained better r esults compared t o 2008. A truly impressive
accomplishment was r epresented by the ability of developing c ountries to serotype several strains
correctly in 2009.
We t hink that the m ain problem in identifying the cor rect s erotype was linked t o difficulties in the
characterization of flagellar antigens, which could be the consequence of a lack of good quality antisera,
since laboratories often correctly identified the O antigen and one of the two flagellar antigens. In other
cases, participating laboratories correctly indentified the O antigen and the flagellar antigen complex, but
incorrectly id entified the minor a ntigens w ithin the c omplex. Our p roposed explanation is further
supported b y t he r esults r eported f or s train WHO S 9.2 ( Brandenburg / I 4,12: l,v:e,n,z15), w hich
accounted for the hi ghest number (19%) of deviations. Six laboratories reported it as S. Kimuenza ( I 1,
4,12, 27:l,v:e,n,x) by wrongly detecting the E-complex, and three laboratories reported it as S. Mons (I 1,
4,12, 27:d:l,v) by incorrectly determining both the first and second flagella phase.
Similar problems were observed with strain WHO S9.4 (Bredeney / I 1,4,12:l,v:1,7); a 1-complex strain
which resulted in 16% of deviations. Four laboratories reported it as S. Fyris (I 4,[5],12:l,v:1,2) because of
incorrect detection of the second flagella phase 1,2.
The strain WHO S 9.6 S. Worthington ( I 1,13, 23:z:l,w) was incorrectly serogrouped b y 10.9 % of
participating laboratories. Additionally, 16.3% of participating laboratories failed to correctly serotype it.
A like ly explanation is that s erogrouping a nd s erotyping t his strain require a us ual p anel of s omatic
antisera; 13 and 23.
4.2 Antimicrobial susceptibility testing (AST) of Salmonella strains

Overall, 94% of the Salmonella AST was correctly performed, and critical deviations were only 3%. This
result is extremely satisfactory and represents the best achievement in the EQAS trials performed through
the different years. Of note, the number of participating laboratories decreased from 168 in 2008 to 153 in
2009. Thus, the obt ained r esults could be due t o l ack of pa rticipation o f laboratories which performed
poorly in previous years. However, the excellent results obtained could also be the consequence of better
14
performance by laboratories p articipating in training cou rses aiming to strengthen awareness about
antimicrobial resistance.
Guidelines for MIC breakpoint interpretation were given to participating laboratories also in EQAS 2009.
In a ddition, expert g uidelines on t he i nterpretation o f cepha losporin resistance w ere also distributed t o
instruct laboratories to report resistance to all cephalosporins regardless of MIC, in case resistance to one
cephalosporin w as observed. Similarly, participating la boratories were asked to utilize E UCAST
epidemiologic br eak-points f or i nterpretation of CIP susceptibility. The EQAS organizers utilized the
lower epidemiologic breakpoint for ciprofloxacin to facilitate the detection of low-level resistance which
may be caused either by alteration of the drug target due to a single point mutation in the gyrase-encoding
gene or by protection of t he dr ug t arget due t o Q nr pr oteins w hich a re e ncoded b y plasmid-mediated
genes. Accurate de tection of t hese low-level ci profloxacin-resistant strains is e ssential to warrant
appropriate clinical treatment. Indeed, patients infected with low-level ciprofloxacin-resistant strains may
have either a hi gher l ikelihood of treatment failure or a poo r c linical r esponse if t reated with
fluoroquinolones. O f not e, l ow-level ci profloxacin-resistant strains would be interpreted as s usceptible
according to current CLSI clinical breakpoints.
Participating la boratories ha d familiarity w ith the int erpretation rules adopted by t he E QAS or ganizers
since t hey w ere given the interpretative guidelines and r ecommendations (see M aterials and Methods
section) in the last two years, which could have contributed to the better performance achieved in 2009.
As in previous years, a high pe rcentage of t otal deviations was observed for CIP, STR, SMX and TET
susceptibility tests. In case of CIP susceptibility test, the participating laboratories obtained less than 90%
correct results for strain WHO S-9.7. This strain had MIC values of 0.12 µg/ml, and therefore should have
been considered resistant ( since it ha d reduced s usceptibility) to c iprofloxacin. Likely, participating
laboratories interpreted the results according to CLSI breakpoints instead of the recommended EUCAST
cut-off values. In case of STR susceptibility test, in EQAS 2009 w e observed difficulties comparable to
what observed in previous EQAS iterations, since many strains had zone diameters o r MIC values near
the br eakpoint. In 2009, less t han 90% of t he p articipants ha d correct i nterpretation in s ix of t he e ight
strains (Table 7) . As a consequence, DTU F ood launched a s tudy a mong 17 l aboratories f rom E urope,
China a nd North America t o establish an exact br eakpoint for r esistance, a nd t he obtained results have
now been submitted to a scientific journal fo r editorial revision. In case of SMX susceptibility te st, we
observed more de viations i n t he r esults r eported in E QAS 2009 t han i n previous EQAS ite rations. The
potency of this antimicrobial is highly dependent on the quality of the test media used for susceptibility
15
testing, a nd it is w ell k nown t hat SMX breakpoints ar e difficult to interpret. The refore, t he observed
deviations could have been caused by high th ymidine a nd th ymine content in the me dium, w hich
antagonize the ef fects o f S MX and / or by difficulties in the int erpretation of s ulfonamide b reakpoints,
since it is common to observe l ight growth i n t he i nhibition halo near t he s ulphonamide breakpoint. Of
importance, sulfonamide z one di ameters s hould be m easured f rom t he p oint of 80% i nhibition and not
from t he poi nt of complete i nhibition which i s typically utilized for interpretation of susceptibility tests
for other cl asses of a ntimicrobials. Although four (50%) of t he strains included in E QAS 2009 were
susceptible to SMX, less than 90% of the laboratories obtained correct results, and they instead classified
these strains as r esistant. Finally, in case o f T ET s usceptibility t est, the o bserved deviations could have
been caused by the sensitivity of this antimicrobial to the pH of Müller Hinton media used or they might
indicate that the CLSI clinical breakpoint should be reconsidered.
In general, data from the Salmonella AST component of EQAS 2009 demonstrate an overall improvement
which c ould be t he consequence of i) p articipation of a de creased num ber of laboratories f rom Central
Asia & Middle E ast compared t o 2008; i i) test st rains typeable m ore easily t han strains pr ovided in
previous E QAS i terations; and iii) improved a wareness c oncerning antimicrobial r esistance also due t o
WHO GF N laboratory training cou rses. Of not e, fewer l aboratories from China, E urope a nd N orth
America and more laboratories from the Southeast Asian and African regions participated to this EQAS
iteration compared to 2008.
When pe rforming AST, the i nclusion of reference s trains f or i nternal QC is e xtremely impor tant. If
correctly used, the reference strain will provide QC for both the method and the reagents. Unfortunately,
only 129 (84%) participating laboratories submitted AST results of the QC strain. We always encourage
laboratories to conduct quality assurance when performing AST and, to facilitate internal QC, we provide
each n ew pa rticipating l aboratory w ith t he reference strain E. coli ATCC 25922. Laboratories
participating in EQAS are invited to retain and maintain the QC strain for future use. As a rule, results for
the test organisms should not be reported if ≥ 3 out of 30 results for the QC strain are outside the expected
interval. Unfortunately, we did not observe any improvement in AST of QC strains by using either disk
diffusion or MIC determination, as a high number of laboratories reported results outside the accepted QC
interval. These erroneous results typically arise from inadequate standardization of methodologies, lack of
good quality culture media and improper s torage of antimicrobial-containing disks. Thus, deviations i n
AST results can likely be corrected by improving QC practices. For example, if the use of cotton swabs
for pl ating ba cteria c auses r epeated failures to obt ain va lues w ithin t he a cceptable Q C i nterval, we
16
recommend dispensing different vol umes of bacterial inoculum ont o M üller H inton II a gar p lates t o
determine the exact volume necessary to obtain acceptable results.
In conclusion, the EQAS 2009 results showed an improvement in Salmonella AST which, however, still
need harmonization. In addition, EQAS aims at improving the component related to AST of the QC strain
which, i n 2009, was l ess s atisfactory than in previous y ears. It is impor tant to emphasize th at thi s
component represents the true indicator of the quality of AST performance.
4.3 Serogrouping and serotyping of Shigella strains

In EQAS 2009, t he component related to Shigella serotyping was available for all regions. Participating
laboratories were scattered in all regions excluding the Caribbean. Of note, between 103 (95%) and 114
(99%) participating laboratories serogrouped the test strains with maximum one deviation for three of the
strains a nd f ive deviations for t he Shigella boydii strain. In a ddition, up t o 70 participating laboratories
serotyped the strains and, as for the serogrouping, the majority of the observed deviations was related to
Shigella boydii typing. Surprisingly, eight laboratories reported the same erroneous serotype (serotype 1).
Need o f improvements were identified mainly i n the African region where eight laboratories pe rformed
Shigella serotyping w ith onl y 72% of correct r esults. The considerable number of l aboratories
participating to this EQAS component indicates that Shigella is an important human pathogen and that the
inclusion of this microorganism in EQAS was a wise decision.
4.4 Antimicrobial susceptibility testing (AST) of Shigella strains

In E QAS 2009, A ST of Shigella spp. was ava ilable f or al l r egions, and was pe rformed by 111
laboratories. A ll regions s ubmitted results w ith the e xception of t he O ceanic r egion, a nd t he o verall
regional pe rformance w as s imilar t o t he on e de scribed f or Salmonella AST. The results r eported f or
Shigella AST revealed similar pr oblems a s described for Salmonella. Accordingly, w e obs erved hi gh
percentages of deviations related to TET a nd STR susceptibility t est r esults. Possible reasons f or these
deviations have already been discussed in section 4.2. SMX and CIP susceptibility test results were not as
deviating as de scribed f or Salmonella. A like ly explanation is r epresented by t he f act t hat all Shigella
strains were susceptible to CIP, and the participants could then avoid misinterpretation of strains having
reduced s usceptibility to CIP. Surprisingly, participating laboratories pe rformed SMX s usceptibility
testing of Shigella more correctly then SMX susceptibility testing of Salmonella.
17
4.5 Identification of Campylobacter strains
In 2009, w e s elected both Campylobacter jejuni (not i ncluded i n E QAS t rials s ince 2006) and
Campylobacter coli strains. To avoid viability problems, the stability of lyophilized cultures was tested by
DTU F ood pr ior t o appoint t he producer, and was c onfirmed b y vi ability testing of the l yophilized
cultures in J anuary 201 0. A large number (34%) of l aboratories r equesting the s trains d id not s ubmit
results, a s i t w as obs erved i n pr evious years. Probably, this represents a lack of c apacity to gr ow
Campylobacter strains which r equire i ncubation i n microaerophilic atmosphere. We did not e xamine i f
this lack of c apacity w as r egional-based, but a nalysis of th e r esults s trongly indi cate tha t participating
laboratories in the African and Central Asian & Middle Eastern region had the worst performance. As a
consequence, t he W HO GFN planned to c onduct a s pecific l aboratory t raining c ourse on i solation of
Campylobacter for E nglish-speaking A frican l aboratories i n 2010 . O verall, t he r esults related to
Campylobacter identification were excellent, and 95% of the submitted results for C. jejuni were correct.
This is the first EQAS iteration in which the percentage of correctly identified Campylobacter strains is
higher than 90%.
4.6 Antimicrobial susceptibility testing (AST) of Campylobacter strains

In EQAS 2009, a Campylobacter AST component was added. However, only data obtained through the
MIC de termination method were ac cepted, s ince international r ecognized di sk di ffusion i nterpretation
guidelines do not exist. In addition, epidemiological cut-off values recommended by EUCAST were used
for A ST i nterpretation, which a llows to categorize s trains in susceptible ( wild-type) or r esistant ( non-
wild-type). The 25 participating laboratories performed satisfactorily, since they obt ained 91.4% correct
test r esults, w hich i s ve ry close t o t he threshold criteria s et f or Salmonella. N o l aboratories from t he
Central A sia & M iddle E astern, C aribbean, Oceanic and Russian r egions pa rticipated in this EQAS
component. The t wo participating laboratories from A frica reported m ore deviating results compared to
laboratories from other regions.
The m ajority o f obs erved deviations was linked t o E RY, N AL, S TR and TET susceptibility testing.
Inconsistent deviations were observed for CIP and NAL susceptibility testing, which is surprising since
resistance to these antimicrobials in Campylobacter is caus ed by target alteration due t o t he s ame poi nt
mutation(s) in gyrA, and therefore similar deviations would be expected. A total of 24 (96%) participating
laboratories submitted AST results for the QC strain. In this case, it was possible to upload data for four
different MIC determination methods, i.e. micro- and agar-dilution performed at two different incubation
conditions (37 °C and 42 °C). The majority of deviations was observed for CIP susceptibility testing b y
micro-dilution at 42 °C and ERY s usceptibility testing by a gar-dilution a t 42 °C. Surprisingly, t he
18
participating laboratories performed better when testing NAL susceptibility for the QC strain than for the
test strains, while in the case of CIP susceptibility testing, they obtained better result for the tests strains
than f or t he Q C s train. In general, AST of the Q C strain was s atisfactory. H owever, ERY a nd GEN
susceptibility testing of the QC strain can be improved.
4.7 Identification of the unknown culture

In E QAS 2009 , we included a Vibrio mimicus strain as w e aim to strengthen t he a bility of di agnostic
laboratories to differentiate different Vibrio species. Of 56 laboratories delivering results, only 27 (48%)
identified the strain completely. In EQAS 2007, we included a Vibrio parahaemolyticus strain which was
tested by 86 laboratories, thus showing that in EQAS 2009 the performance of this component was poor
probably due to low viability of the strain.
5. Conclusions
The acceptance t hreshold for t he Salmonella serotyping EQAS component was m et b y 69% (n=105) of
the participating la boratories. In addition, 83% of t he laboratories te sted a ll e ight strains and a t otal of
86% of a ll tests were co rrect, t hus r epresenting an i ncrease c ompared t o 2008. H owever, t he ability in
testing correctly the i nternal QC strain decreased of 3% compared t o 2008. M any o f t he r egions
performed satisfactorily, with a r esult overall similar to last year. However, the Salmonella serotyping
capacity of l aboratories i n African a nd C entral A sian & Middle E astern regions s till needs to be
improved. Future training efforts should aim at enhancing the capability to detect the flagella phases, and
at distributing protocols for preparing hi gh quality swarm agar plates. The obtained results indicate that
detection of the phase two flagellar antigen is the most critical point for obtaining satisfactory serotyping
results. In addition, these results show that many laboratories in developing countries still need supplies of
antisera to facilitate serotyping of strains with rare antigenic formulae.
Concerning the Salmonella AST component, the obtained results emphasize the importance to harmonize
the m ethodology and to provide adequate g uidelines. Indeed, analysis o f the results indicate that the
distribution of the la test guidelines for breakpoint interpretation and t he s trengthened awareness of the
importance of performing an internal Q C h ave i ncreased the ability of most la boratories to perform
correct A ST. Overall, the acc eptance t hreshold w as m et, a nd w e identified 3 m inor a nd 3 c ritical
deviations. Notably, STR, SMX, CIP and TET caus ed the majority of the observed deviations as in the
previous EQAS iterations. No regional underperformance was observed, and the Central Asian & Middle
Eastern regions improved considerably compared to EQAS 2008. Unfortunately, 24 (16%) participating
19
laboratories di d not r eport da ta f or AST of the Q C s train despite the E QAS or ganizers repeatedly
recommended the use of such QC strains and are willing to provide them. Once more, we want to remind
the importance of the use of QC strains for optimizing the methodology in use, since many laboratories
reported values out of the accepted QC range both for MIC determination and for disk diffusion.
A Shigella component was included also in EQAS 2009, a nd consisted of serogrouping, s erotyping and
AST. Most laboratories (n=103; 87%) correctly serogrouped the four Shigella strains, and a maximum of
4.6% de viations was ob served. A t otal of 70 laboratories p erformed s erotyping, with a ma ximum of
12.5% deviations. Only minor regional differences were observed, and the highest number of deviations
was reported from laboratories from the African region.
The results obt ained i n t he Shigella AST c omponent suggest c onclusions s imilar to the ones r eported
above concerning the Salmonella AST.
A total of 131 l aboratories requested to participate to the Campylobacter component of EQAS 2009, but
only 86 (66%) uploaded data related to identification. The C. jejuni strain was correctly identified by 95%
of the p articipating la boratories. The ma jority o f di fficulties in Campylobacter identification were
experienced by laboratories i n the African and Central A sian & M iddle E astern r egions. Therefore, a
laboratory training course on Campylobacter identification has been scheduled in Africa in 2010.
EQAS 2 009 i ncluded a n A ST component for Campylobacter, where only MIC de terminations w ere
considered acceptable. A total of 25 laboratories participated to this component. The acceptance threshold
used for Salmonella was appl ied and was al most m et, since we obs erved 0.7% m inor and 8.6% c ritical
deviations. T he da ta r evealed t hat E RY, N AL, S TR a nd T ET s usceptibility testing were the m ost
challenging. In addition, discrepancies between NAL and CIP susceptibility testing were observed. Of the
25 participating laboratories, 24 performed AST of the QC strain, and the majority of the results for ERY
and GEN susceptibility was out of the accepted range.
The unknown strain, Vibrio mimicus, was identified by 75% of the participating laboratories at the genus
level (Vibrio spp.), and by 48% of the participating laboratories at the species level (V. mimicus).
20
Reference List
1. Clinical a nd L aboratory S tandards I nstitute. M ethods f or Antimicrobial D ilution a nd Disk S usceptibility T esting o f
Infrequently Isolated or Fastidious Bacteria. M45-A. 2006. Wayne, PA, USA.
2. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk and Dilution Susceptibility
Tests for bacteria Isolated from Animals. M31-A3. 3rd Edition[Approved Standard]. 2008. Wayne, PA, USA.
3. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. M100-S19.
Nineteenth Informational Supplement. 2009. Wayne, PA, USA.
4. Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility T ests for Bacteria T hat
Grow Aerobically. M07-A7. 7th Edition[Approved Standard]. 2006. Wayne, PA, USA.
5. Grimont, P . A . D . Antigenic f ormulae o f t he Salmonella serovars. F .X.Weil. [9th e d.]. 2 007. Paris, F rance., W HO
Collaborating Center for Reference and Research on Salmonella, Institut Pasteur.
6. Herikstad, H ., Y . Motarjemi, an d R . V . T auxe. 2 002. Salmonella surveillance: a g lobal s urvey o f p ublic health
serotyping. Epidemiol.Infect. 129(1):1-8.
7. Schwarz, S., P. Silley, S. Simjee, N. Woodford, D. E. van, A. P. Johnson, and W. Gaastra. 2010. Editorial: assessing the
antimicrobial susceptibility of bacteria obtained from animals. J.Antimicrob.Chemother. 65(4):601-604.
21
Figure and Tables
Figure 1. Countries participating* in the WHO EQAS 2009
*marked in yellow
22
Table 1. EQAS participating laboratories’ performance of Salmonella serotyping
EQAS Labs serotyping all

Correct test results
iteration provided strains
No. % No. %
2000 34 92 165 76
2001 79 82 513 72
2002 80 81 668 91
2003 69 54 692 80
2004 78 61 701 81
2006 105 81 808 85
2007 109 78 920 88
2008 100 66 888 83
2009 119 83 974 86
Average 86 75 703 82
Table 2. Ability of EQAS participating laboratories to serotype the test Salmonella strains
Number Participating laboratories

of strains EQAS EQAS EQAS EQAS EQAS
correctly 2000 2001 2002 2003 2004
serotyped
No. % No. % No. % No. % No. %
8 9 24 34 35 52 53 32 25 41 32
7 9 24 13 14 19 19 15 12 14 11
6 4 11 9 9 12 12 18 14 16 13
5 3 8 9 9 4 4 23 18 16 13
4 3 8 4 4 1 1 14 11 11 9
3 4 11 8 8 4 4 13 10 10 8
2 2 5 3 3 5 5 4 3 10 8
1 2 5 5 5 1 1 5 4 5 4
0 1 3 11 11 1 1 3 2 4 3
In total 37 100 96 100 99 100 127 100 127 100
Number Participating laboratories

of strains AVERAGE
EQAS EQAS EQAS EQAS
correctly EQAS
2006 2007 2008 2009 2000 - 2009
serotyped
No. % No. % No. % No. % No. %
8 42 32 66 47 50 33 76 50 44 37
7 35 27 29 21 36 24 29 19 20 18
6 19 15 13 9 11 7 7 5 12 11
5 12 9 11 8 14 9 13 8 11 10
4 7 5 7 5 12 8 5 3 7 6
3 5 4 6 4 9 6 7 5 7 7
2 3 2 2 1 8 6 5 3 4 4
1 4 3 6 4 9 6 6 4 4 4
0 3 2 0 0 2 1 5 3 3 3
In total 130 100 140 100 151 100 153 100 114 100
23
Table 3. Region-based categorization of EQAS participants’ performance of Salmonella serotyping
No. of % strains
EQAS No. of Countries participating
Region strains correctly
iteration labs in EQAS 2009
serotyped serotyped
2001 6 37 73.0
2002 9 62 87.1
2003 11 70 71.4 Algeria, Cameroon, Central African
2004 9 51 62.7 Republic, Democratic Republic of
Africa Congo, Gambia, Ivory Coast, Kenya,
2006 16 95 71.6 Madagascar, Mauritius, Morocco,
2007 11 73 80.8 South Africa, Tunisia
2008 10 71 49.3
2009 15 94 75.5
2001 10 60 50.0
2002 5 30 83.3
2003 5 35 54.3
Asia & Middle 2004 5 33 54.5
Egypt, Israel, Jordan, Oman, Yemen
East 2006 5 35 74.3
2007 5 40 55.0
2008 5 34 61.8
2009 5 32 46.9
2001 0 0 0
2002 0 0 0
2003 3 18 61.1
2004 2 8 87.5 Barbados, Suriname, Trinidad and
Caribbean
2006 3 14 78.6 Tobago
2007 2 9 77.8
2008 3 14 78.6
2009 3 12 83.3
2001 4 32 96.9
2002 3 24 100.0
2003 8 60 75.0
2004 7 46 78.3
China China
2006 6 48 85.4
2007 10 80 91.3
2008 15 108 94.4
2009 16 126 95.2
2001 43 323 80.5
Albania, Belgium, Bosnia and
2002 50 384 90.0 Herzegovina, Bulgaria, Croatia,
2003 60 401 84.8 Cyprus, Czech Republic, Denmark,
2004 57 392 84.7 Estonia, Finland, France, Germany,
Europe Greece, Hungary, Ireland, Italy,
2006 52 403 86.4 Lithuania, Luxembourg, Malta,
2007 54 415 89.4 Republic of Moldova, Poland,
2008 50 379 82.3 Serbia, Slovak Republic, Slovenia,
Turkey, United Kingdom
2009 47 362 93.1
24
Table 3 (continued). Region-based categorization of EQAS participants’ performance of Salmonella serotyping
No. of % strains
EQAS No. of Countries participating
Region strains correctly
iteration labs in EQAS 2009
serotyped serotyped
2001 4 32 87.5
2002 2 16 100.0
2003 6 41 95.1
2004 8 55 81.8
North America Canada, United States of America
2006 10 80 96.3
2007 12 94 97.9
2008 11 84 95.2
2009 12 90 92.2
2001 4 30 100.0
2002 6 43 93.0
2003 6 46 93.5
2004 5 38 97.4
Oceania Australia,New Zealand
2006 5 37 94.6
2007 4 32 100.0
2008 4 30 93.3
2009 4 32 96.9
2001 1 8 12.5
2002 1 8 62.5
2003 1 7 14.3
2004 4 26 69.2
Russia Belarus, Georgia, Russia
2006 5 40 80.0
2007 8 51 80.4
2008 6 40 90.0
2009 7 49 91.8
2001 11 78 57.7
2002 11 82 87.8
2003 13 83 75.9 Argentina, Bolivia, Brazil, Chile,
88 79.5 Colombia, Costa Rica, Cuba,
2004 15
Latin America Ecuador, Guatemale, Honduras,
2006 13 84 84.5
Mexico, Nicaragua, Paraguay, Peru,
2007 15 107 88.8 Venezuela
2008 17 120 71.7
2009 21 150 77.3
2001 15 113 54.0
2002 12 90 92.2
2003 15 100 81.0 Brunei, Cambodia, Japan, Lao,
2004 17 130 81.5 Malaysia, Philippines, Singapore,
Southeast Asia
2006 15 117 84.6 South Korea, Sri Lanka, Taiwan,
2007 19 140 91.4 Thailand, Vietnam
2008 18 125 81.6
2009 23 180 81.1
25
Table 4. Salmonella serogroups (SG), serotypes (ST) and deviations (D), WHO EQAS 2009
Strain Correct serotype No. of labs % DSG No. of labs % DST Deviating results (*)
ID reporting reporting
SG ST
Dublin (3), Nitra (1), Moscow (1),
WHO
Enteritidis 9,12:g,m:- 159 3.1 151 6.6 Blegdam (1), Stanley (1), Rostock (1),
S-9.1 Onarimon (1), Paratyphi A (1)
Kimuenza (6), Mons (3),
Typhimurium (2), Sandiego (2), Essen(1),
Fyris (1), Texas (1), Azteca (1),
WHO
Brandenburg 4,12:l,v:e,n,z15 157 1.3 142 19.0 Kisangani (1), Reinickendorf (1),
S-9.2 Wilhelmsburg (1), Tsevie (1),
Brezany (1), Koessen (1), Wagenia (1),
Chartres (1), Duisburg (1), II (1)
Vejle (2), Newlands (2), Lamberhurst (2),
WHO Lamin (1), Ratchaburi (1), Aminatu (1),
Muenster 3,10:e,h:1,5 152 3.3 142 12.7
S-9.3 Vilvoorde (1), Lorochelle (1),
Sekondi(1), II (1), Anatum (1),
Fyris (4), Brandenburg (3), Saintpaul (2),
Give (1), Hato (1), Togo (1),
Schwarrzengrund (1), Typhimurium (1),
WHO
Bredeney 1,4,12:l,v:1,7 157 3.8 144 16.0 Azteca (1), Svedvi (1), Concord (1),
S-9.4 Kubacha (1), Reading (1), Stanley (1),
Kaapstad (1), Parkroyal (1),
II 1,9,12:l,w:e,n,z
Chester (8), Saintpaul (4), Reading (2),
WHO Chartres (2), Typhimurium (2),
Sandiego 4,5,12:e,h:e,n,z15 157 0.6 142 14.8
S-9.5 Duisburg (1), Brandenburg (1),
Arechavaleta (1)
Carno (3), Nanga (3), Tanzania (2),

Gabon (1), Poona (1), Vridi (1),
WHO Paratyphi A (1), Enteritidis (1),
Worthington 1,13,23:z:l,w 147 10.9 129 16.3
S-9.6 Wortington (1), Washington (1),
Remiremont (1), Ajiobo (1), Alkmaar (1),
Koessen (1), Marburg (1), II 1,13,23:z:1,5
Corvallis (3), Altona (2), Tallahassee (2),

WHO Dabou (1), Cocody (1), Kalamu (1),
Albany 8,20:z4,z24:- 155 2.6 134 10.4
S-9.7 Sindelfingen (1), Manhattan (1),
Blockley (1),Yovokome (1)
Typhimurium (5), Duisburg (3),

Schwarzengrund (3), Fyris (1), Typhi (1),
WHO
Stanley 4,5,12:d:1,2 156 0.6 143 13.3 Clackamas (1), Saintpaul (1), Paratyphi B
S-9.8 var. Java (1), Paratyphi B (1),
Eppendorf (1), Ayinde (1)
*number of participants reporting the specified deviating result
26
Table 5. EQAS participating laboratories’ performance of internal quality control strain (WHO
S-9.1, Salmonella Enteritidis) serotyping
EQAS Labs serotyping

iteration S. Enteritidis correctly
No. %
2000 34 92
2001 64 84
2004 113 95
2006 116 94
2007 135 96
2008 139 96
2009 141 93
Average 106 93
27
Table 6. EQAS participating laboratories’ performance of antimicrobial susceptibility testing of Salmonella strains
EQAS No. of EQAS Average no. of % correct test % minor % major % very major % critical % total deviations
iteration participating antimicrobial results deviations deviations deviations deviations (S → R & R → S &
laboratories agents tested (S ↔ I or I ↔ R)^ (S → R)^ (R→ S)^ (R→ S & S → R)^ S ↔ I or I ↔ R)^
2000 44 9.1 92 4 4 0 4 8
2001 108 8.9 91 6 2 1 3 9
2002 119 8.9 92 6 2 1 3 9
2003* 147 8.1 93 4 3 0 3 7
2004 152 10.2 93 4 2 1 3 7
2006 143 11.2 88 8 3 1 4 12
2007 143 10.8 93 4 2 1 3 7
2008 168 10.3 91 4 2 3 5 9
2009 153 10.4 94 3 2 1 3 6
Average* 131 9.8 92 5 2 1 3 8
*Data do not include one strain which may have lost resistance due to transport or storage stress
^S, susceptible; I, intermediate; R, resistant
Table 7. Antimicrobial susceptibility test results (number of R/I/S) for the EQAS 2009 Salmonella strains*
Strain Antimicrobial^
AMP CTX CAZ CRO CHL CIP
STR GEN
SMX NAL
SXT TET TMP
WHO
10/10/132 3/2/121 1/2/113 0/0/98 2/1/137 0/1/149 142/0/2 1/0/138 104/1/4 76/1/3 5/0/126 14/19/105 1/1/75
S-9.1
WHO
6/3/142 1/1/124 0/0/115 0/2/96 1/0/138 0/2/147 4/2/137 1/3/134 9/33/65 10/3/65 4/2/125 1/9/130 2/0/75
S-9.2
WHO
5/4/142 1/1/124 0/0/115 0/0/98 2/1/135 0/1/148 1/1/141 2/4/131 9/16/83 12/1/65 6/1/122 6/14/120 1/0/77
S-9.3
WHO
3/2/146 1/0/125 0/1/114 0/1/97 1/0/138 1/1/147 2/3/138 1/3/133 11/21/76 7/1/70 4/1/127 4/7/129 1/0/75
S-9.4
WHO
4/2/144 1/0/124 0/0/115 0/0/97 1/0/137 1/2/145 1/3/138 1/3/133 6/31/71 11/3/63 4/1/125 5/8/125 1/0/76
S-9.5
WHO
5/2/143 1/1/124 0/1/114 1/1/94 3/0/135 0/2/146 1/5/136 3/3/130 104/0/4 75/1/2 122/1/6 135/0/3 77/0/0
S-9.6
WHO
147/0/3 1/2/124 0/1/114 0/0/94 136/1/1 45/7/97 5/1/137 130/1/4 23/49/36 77/0/1 129/0/1 132/5/3 76/0/1
S-9.7
WHO
5/3/143 1/1/125 0/0/115 0/0/96 134/1/3 0/1/147 3/2/138 3/3/131 11/34/63 77/0/0 126/1/3 124/12/4 77/0/0
S-9.8
^For antimicrobial abbreviations: see List of Abbreviations page 1

*In bold: expected interpretation. Grey cell: <90% of laboratories did correct interpretation. R, resistant; I, intermediate; S, susceptible.
29
Table 8. EQAS participants’ performance of Salmonella strains antimicrobial susceptibility testing categorized by antimicrobial
EQAS No. of Antimicrobial∞

Performance
iteration labs AMC AMP CAZ CHL CIP POD CRO CTX GEN KAN NAL SMX STR SXT TET TMP XNL OVERALL
No. of tests - 343 - 343 334 - 343 312 328 248 312 - 335 295 - 3193
2000 44 % critical deviations* - 6 - 4 1 - 4 4 1 3 4 - 6 1 - 3
% total deviations^ - 8 - 7 6 - 5 16 4 5 12 - 13 1 - 8
No. of tests - 822 - 814 813 - 821 623 726 431 679 757 804 416 - 7706
2001 108 % critical deviations* - 4 - 2 1 - 2 2 2 6 7 2 7 1 - 3
% total deviations^ - 7 - 3 4 - 4 7 8 9 27 5 18 2 - 9
No. of tests - 918 - 903 911 - 905 680 885 495 718 724 861 499 - 8499
2002 119 % critical deviations* - 2 - 2 0 - 2 2 2 4 4 7 3 3 - 3
No. of tests - 1019 - 996 995 - 993 738 947 615 768 929 995 582 - 9577
2003● 147 % critical deviations* - 2 - 1 0 - 2 2 1 4 9 2 4 1 - 3
No. of tests 973 1178 - 1159 1162 - - 995 1201 - 1130 734 947 1051 1122 729 - 12381
2004 152 % critical deviations* 6 3 - 2 0 - - 0 2 - 1 5 1 3 5 2 - 3
% total deviations^ 12 5 - 2 1 - - 14 3 - 4 8 21 4 11 2 - 7
No. of tests 950 1092 769 1060 1110 305 - 956 1078 - 1035 649 896 996 1054 607 225 12782
2006 143 % critical deviations* 9 2 7 3 2 1 - 7 3 - 2 6 5 3 9 1 2 4
% total deviations^ 22 3 11 15 6 26 - 15 7 - 6 7 22 5 20 2 9 12
No. of tests 908 1114 830 1105 1101 389 - 914 1111 - 1092 678 875 971 1047 583 258 12976
2007 143 % critical deviations* 6 5 1 0 1 4 - 1 3 - 2 5 4 3 4 1 0 3
% total deviations^ 17 7 1 6 1 16 - 2 4 - 3 6 26 3 11 2 6 7
No. of tests - 1331 961 1226 1307 - 791 1104 1265 - 1168 718 867 1155 1249 696 - 13858
2008 168 % critical deviations* - 3 3 1 19 - 3 3 4 - 2 4 7 3 6 2 - 5
% total deviations^ - 8 6 11 21 - 6 6 6 - 4 5 25 4 13 2 - 9
No. of tests - 1206 921 1108 1190 - 775 1009 1143 - 1095 624 864 1042 1114 616 - 12707
2009 153 % critical deviations* - 3 1 1 8 - 0 1 2 - 1 7 9 3 4 1 - 3
No. of tests - 9023 3481 8714 8923 - 1566 4978 8860 - 8406 5192 6926 7625 8581 5023 - 87298
Average● 1531 % critical deviations* - 3 3 2 5 - 2 3 3 - 1 5 5 3 5 1 - 3
30
Legend Figure 8
∞
For antimicrobial abbreviations: see List of Abbreviations page 1
*R→ S & S → R (R, resistant; S, susceptible)
^S→R & R→S & S↔I or I↔R (I, intermediate)
●
Data do not include one strain which may have lost resistance due to transport or storage stress
-, not determined
31
Table 9. Region-based categorization of EQAS participants’ performance of Salmonella antimicrobial susceptibility testing
Region EQAS No. % correct % minor % major % very % critical % total Countries participating
iteration of test deviations deviations major deviations deviations in the 2009 iteration
labs result (S ↔ I or (S → R)^ deviations (S → R & (S→R & R→S
I ↔ R)^ (R → S)^ R → S)^ & S↔I or
I↔R)^
2001 7 80.1 9.6 7.7 2.5 10.2 19.8 Algeria, Cameroon,
2002 10 94.3 4.1 1.0 0.6 1.6 5.7 Central African Republic,
2003 13 86.9 6.6 2.8 3.7 6.5 13.1 Democratic Republic of
Congo, Ethiopia, Gambia,
Africa
2004 11 85.7 7.2 5.2 1.9 7.1 14.3

Ivory Coast, Kenya,
2006 20 85.8 7.5 4.1 2.7 6.8 14.3 Madagascar, Mauritius,
2007 16 90,7 4.4 4.0 0.9 4.9 9.3 Morocco, Namibia,
2008 19 83.8 6.5 5.5 4.2 9.7 16.2 Nigeria, South Africa,
Sudan, Tunisia
2009 22 90.1 4.5 3.6 1.8 5.4 9.9
2001 10 87.7 6.3 5.2 0.8 6.0 12.3
2002 6 83.4 9.8 6.6 0.2 6.8 16.6
Central Asia &
8 89.9 4.5 4.0 1.6 5.6 10.1

Middle East
2003
2004 10 87.5 6.7 5.5 0.3 5.8 12.5 Egypt, Iran, Israel, Jordan,
2006 7 79.2 10.5 9.8 0.5 10.3 20.8 Oman, Yemen
2007 8 87.8 5.0 6.2 1.1 7.3 12.2
2008 12 86.1 6.5 4.0 3.4 7.4 13.9
2009 6 93.7 4.3 0.9 1.1 2.0 6.3
2001 2 83.5 9.5 7.0 0.0 7.0 16.5
2002 1 95.8 4.2 0.0 0.0 0.0 4.2
2003 8 91.7 6.4 1.5 0.5 2.0 8.4
Caribbean
Barbados, Jamaica,
2004 8 94.1 3.1 1.9 0.9 2.8 5.9
Suriname, Trinidad and
2006 5 92.1 5.4 1.6 1.0 2.6 8.0 Tobago
2007 4 95.0 3.1 0.9 0.9 1.8 5.0
2008 5 90.7 5.5 0.9 2.9 3.8 9.3
2009 4 93.2 1.8 3.2 1.8 5.0 6.8
2001 4 98.9 0.8 0.0 0.3 0.3 1.1
2002 3 96.0 4.0 0.0 0.0 0.0 4.0
2003 8 90.1 3.6 2.8 3.6 6.4 10.0
China
2004 8 96.0 3.2 0.7 0.1 0.8 4.0

China
2006 6 89.6 7.0 2.9 0.5 3.4 10.4
2007 10 98.3 1.1 0.3 0.2 0.5 1.6
2008 18 92.8 3.7 0.8 2.7 3.5 7.2
2009 14 94.8 2.2 2.1 0.8 2.9 5.1
2001 47 91.3 5.7 2.7 0.3 3.0 8.7 Albania, Belgium, Bosnia
2002 57 92.7 5.2 1.2 0.9 2.1 7.3 and Herzegovina, Bulgaria,
Croatia, Denmark, Finland,
2003 64 92.9 3.8 1.0 2.3 3.3 7.1 France, Greece, Hungary,
Europe
2004 58 93.5 4.3 1.4 0.8 2.2 6.5 Ireland, Italy, Lithuania,
2006 54 88.7 7.0 3.8 0.6 4.4 11.3 Luxembourg, Malta,
Republic of Moldova,
2007 49 94.2 3.7 1.6 0.4 2.0 5.7 Poland, Serbia, Slovak
2008 51 91.2 4.4 2.5 1.9 4.4 8.8 Republic, Slovenia,
2009 40 95.1 2.6 1.3 0.9 2.2 4.8 Turkey, United Kingdom
32
Table 9 (continued). Region-based categorization of EQAS participants’ performance of Salmonella antimicrobial susceptibility testing
Region EQAS No. % correct % minor % major % very % critical % total Countries participating
iteration of test result deviations deviations major deviations deviations in the 2009 iteration
labs (S ↔ I or (S → R)^ deviations (S → R & (S→R & R→S
I ↔ R)^ (R → S)^ R → S)^ & S↔I or
I↔R)^
4
2001 95.8 3.8 0.0 0.4 0.4 4.2
3
2002 90.5 6.9 0.6 2.0 2.6 9.5
North America
7
2003 93.4 5.2 0.0 1.4 1.4 6.6
9
2004 94.2 4,2 1.8 0.0 1.8 6.0 Canada, United States
8
2006 94.8 2.9 1.0 1.3 2.3 5.2 of America
10
2007 95.4 2.9 0.8 0.8 1.6 4.6
14
2008 96.4 0.6 0.4 2.6 3.0 3.6
2009 10 98.7 0.0 0.4 0.9 1.3 1.3
2001 6 91.8 4.7 2.7 0.9 3.6 8.2
2002 7 91.7 6.2 0.0 2.0 2.0 8.3
2003 9 94.3 2.5 1.2 2.0 3.2 5.7
Oceania
2004 11 97.1 2.5 0.3 0.1 0.4 2.9

Australia, New Zealand
2006 7 93.4 4.6 0.9 1.1 2.0 6.6
2007 1 98.9 1.1 0.0 0.0 0.0 1.1
2008 4 93.9 3.8 0.0 2.3 2.3 6.1
2009 4 95.9 3.2 0.3 0.6 0.9 4.1
2001 1 81,9 15,3 2,8 0.0 2.8 18.1
2002 1 84,5 9,9 5,6 0.0 5.6 15.5
2003 1 100.0 0.0 0.0 0.0 0.0 0.0
91.2 6.6 1.5 0.7
Russia
2004 4 2.2 8.8 Belarus, Georgia,

2006 5 87.4 8.2 2.7 1.7 4.4 12.6 Russia
2007 8 88.9 5.8 4.8 0.4 5.2 11.0
2008 6 92.2 4.7 1.4 1.7 3.1 7.8
2009 6 93.8 2.1 3.3 0.8 4.1 6.2
2001 11 90.8 6.9 1.4 1.0 2.4 9.2
93.7 4.6 0.7 1.0 Argentina, Bolivia,
2002 13 1.7 6.3
Brazil, Chile, Colombia,
South America
2003 12 90.8 4.2 2.0 3.0 5.0 9.2 Costa Rica, Cuba,
2004 17 94.4 4.7 0.8 0.1 0.9 5.6 Ecuador, Guatemale,
2006 16 88.7 6.3 4.5 0.6 5.1 11.3 Honduras, Mexico,
17 94.9 1.8 1.9 1.4 3.3 5.0 Nicaragua, Panama,
2007
Paraguay, Peru,
2008 20 93.0 3.4 1.5 2.1 3.6 7.0 Uruguay, Venezuela
2009 20 95.6 2.1 1.1 1.2 2.3 4.4
2001 16 88.1 7.7 2.3 1.9 4.2 11.9
2002 18 89.0 8.1 1.4 1.6 3.0 11.0 Brunei, Cambodia,
Southeast Asia
2003 17 87.4 5.2 4.7 2.7 7.4 12.6 India, Japan, Lao,
Malaysia, Nepal,
2004 16 92.8 4.4 2.3 0.5 2.8 7.2
Philippines, South
2006 15 90.0 8.1 1.2 0.8 2.0 10.0 Korea, Sri Lanka,
2007 20 93.9 4.0 1.4 0.7 2.1 6.1 Taiwan, Thailand,
2008 19 90.5 4.7 2.2 2.6 4.8 9.5 Vietnam
2009 27 91.8 4.1 3.0 1.2 4.2 8.3
33
Table 10. EQAS participants’ performance of antimicrobial susceptibility testing of quality control strain Escherichia coli ATCC 25922
Method Labs' Antimicrobial0
perfor-
AMC AMP CAZ CHL CIP POD CRO CTX ENR FFN2 FIS GEN NAL SMX STR SXT TET TMP XNL
mance4,5
0.004- 0.25- 0.03- 0.03- 0.008 0.004-
Accepted MIC (μg/ml) 2-8 2-8 0.06-0.5 2-8 2-8 0.25-1 1-4 8-32 4-16
3
≤0.5/9.5 0.5-2 0.5-2 0.25-1
0.016 1 0.12 0.12 -0.03 0.015
interval1 Disks (mm) 8-24 16-22 25-32 21-27 30-40 23-28 29-35 29-35 32-40 22-28 15-23 19-26 22-28 15-23 12-20 23-29 18-25 21-28 26-31
2000 No.4 - 37 - 38 35 - - - - - - 39 37 19 36 - 42 31 -
MIC & Disk
(44) %5
- 27 - 37 20 - - - - - - 23 35 53 22 - 42 30 -
2001 No. 4
- 97 - 97 97 - - - - - - 99 74 53 81 90 96 50 -
MIC & Disk
(107) %5
- 19 - 20 14 - - - - - - 12 14 34 12 14 22 22 -
2002 No. 4
- 109 - 107 108 - - - - - - 108 102 57 82 102 102 66 -
MIC & Disk
(114) %5
- 16 - 15 14 - - - - - - 12 14 26 11 12 13 11 -
No. 4
- 140 - 137 138 - - - - - - 138 132 82 105 129 137 79 -
EQAS iteration (total no. of participants)
2003 MIC & Disk

(144) %5 - 14 - 22 9 - - - - - - 9 16 17 9 14 19 14 -
2004 No. 4
117 132 - 128 132 - - 111 - - - 134 126 84 110 120 129 87 -
MIC & Disk
(140) %5
13 10 - 13 8 - - 18 - - - 10 9 16 6 11 13 9 -
2006 No. 4
116 133 96 126 127 39 - 115 19 - - 131 122 74 106 122 125 74 32
MIC & Disk
(137) %5
9 14 15 18 8 12 - 21 63 - - 14 20 29 11 19 12 17 22
2007 No. 4
102 124 92 123 121 47 - 104 - 13 - 124 120 64 97 107 117 67 35
MIC & Disk
(126) %5
8 11 9 14 12 9 - 16 - 0 - 6 7 22 6 13 7 10 11
No. 4
- 147 111 135 144 - - 124 - - 71 145 136 - 101 129 139 79 -
MIC & Disk
%5 - 12 9 10 8 - - 14 - - 14 8 8 - 12 13 7 13 -
2008 No. 4
- 33 23 24 33 - - 23 - - 18 31 23 - 19 22 28 16 -
(147) MIC
%5 - 0 5 0 6 - - 9 - - 11 0 0 - 11 9 0 13 -
No.4 - 114 89 112 111 - - 101 - - 53 114 113 - 82 107 111 63 -
Disk
%5 - 16 10 12 8 - - 15 - - 15 11 10 - 12 14 9 13 -
No.4 - 128 100 121 124 - 88 107 - - 63 123 117 - 98 113 122 70 -
MIC & Disk
% 5
- 16 13 15 7 - 16 10 - - 11 18 13 - 10 14 14 11 -
2009 No.4 - 27 19 24 26 - 20 20 - - 14 25 24 - 19 21 27 25 -
(129) MIC
%5 - 11 11 8 8 - 15 15 - - 21 12 8 - 5 19 11 13 -
No.4 - 101 81 97 98 - 68 87 - - 49 98 93 - 79 92 95 55 -
Disk
%5 - 16 14 16 6 - 16 9 - - 10 18 14 - 11 12 15 11 -
34
Legend table 10
0
1
CLSI standard, Performance Standards for Antimicrobial Disk and Dilution Susceptibility testing. 19th Informational
supplement. CLSI document M100-S19, Wayne, Pennsylvania
2
CLSI s tandars, Performance S tandards f or A ntimicrobial Disk and D ilution S usceptibility Tests f or bacteria Isolated
from Animals. M31-A3. 3rd Edition[Approved Standard]. 2008. Wayne, PA, USA
3
Quality control range developed by the manufacturer of Sensititre
4
No., number of labs performing the analysis
5
%, percentage of labs reporting erroneous results
-, not determined
35
Table 11. Shigella serotypes (ST) and deviations (D), WHO EQAS 2009
Strain Correct No. of labs D (%) Deviating No. of labs D (%) Deviating
serotype reporting results (*) reporting results (*)
correct correct ST
identification
WHO
S. sonnei 114 0.9 (1) N/A N/A N/A
SH-9.1
WHO S. flexneri 2a (1), 2b (1),

112 0.9 (1) 70 5.4
SH-9.2 serotype 6 4a (1)
WHO S. flexneri 6 (1), 4a (1),

111 0.9 (1) 68 4.2
SH-9.3 serotype 2a var Y (1)
WHO S. boydii
103 4.6 (5) 56 12.5 1 (8)
SH-9.4 serotype 2
*number of participants reporting deviating result
36
Table 12. Region-based categorization of laboratories performing Shigella serotyping in 2009
Region No. of No. of strains Strains serotyped Countries participating in the 2009 iteration
laboratories serotyped correctly (%)
Cameroon, Central African Republic, Ethiopia, Gambia, Kenya, Mauritius, South Africa,
Africa 8 18 72.2
Tunisia
Asia & Middle East 3 5 100.0 Israel, Oman, Yemen
Caribbean 0 0 0 -
China 13 35 100.0 China
Albania, Belgium, Czech Republic, Denmark, Finland, Ireland, Italy, Lithuania,
Europe 15 40 92.5
Luxembourg, Republic of Moldova, Serbia, Slovenia, Turkey, United Kingdom
North America 7 18 100.0 Canada, United States of America
Oceanic 3 8 100.0 Australia, New Zealand
Russia 6 18 83.3 Belarus, Georgia, Russia
Argentina, Brazil, Chile, Colombia, Costa Rica, Cuba, Ecuador, Honduras, Mexico,
Latin America 16 40 97.5
Nicaragua, Paraguay, Peru, Uruguay, Venezuela
Southeast Asia 11 30 90.0 Japan, Lao, Philippines, South Korea, Sri Lanka, Taiwan, Thailand
Table 13. EQAS participating laboratories’ performance of Shigella strains antimicrobial susceptibility testing
EQAS iteration No. of % correct test % minor % major % very major % critical % total
participating results deviations deviations deviations deviations deviations
laboratories (S ↔ I or I ↔ R)^ (S → R)^ (R → S)^ (S → R & R → S )^ (S → R & R → S &
S ↔ I or I ↔ R)^
2008 15 95 2 2 1 3 5
2009 111 96 2 1 1 2 4
37
Table 14. Antimicrobial susceptibility test results (number of R/I/S) for the EQAS 2009 Shigella strains*
Strain Antimicrobial∞
AMP CTX CAZ CRO CHL CIP GEN NAL STR SMX SXT TET TMP
WHO
105/0/1 1/0/93 0/0/90 0/1/76 1/0/96 0/0/107 2/1/96 0/1/96 70/0/3 53/0/0 96/1/1 78/14/5 54/0/0
SH-9.1
WHO
4/4/98 1/0/91 0/0/90 0/0/75 1/0/96 0/2/105 2/1/97 1/2/94 11/33/29 3/0/48 2/2/94 1/1/95 2/0/51
SH-9.2
WHO
100/1/4 2/0/90 1/0/87 1/0/72 82/10/4 0/1/105 4/2/93 0/1/95 71/0/1 51/0/1 93/1/2 90/2/3 54/0/0
SH-9.3
WHO
100/1/1 0/0/90 0/0/86 0/0/72 2/0/92 1/0/101 2/0/92 1/0/93 67/2/2 47/0/4 4/1/87 90/1/2 2/0/51
SH-9.4
∞
*In bold: expected interpretation. Grey cell: <90% of laboratories did correct interpretation. R, resistant; I, intermediate; S, susceptible.
Table 15. EQAS laboratories’ performance of Shigella strains antimicrobial susceptibility testing categorized by antimicrobial
EQAS No. of Lab Antimicrobial

iteration labs performance AMP CAZ CHL CIP CTX GEN NAL SMX STR SXT TET TMP CRO OVERALL
No. of tests 52 44 51 48 48 50 52 7 27 52 52 4 42 529
2008 15 % critical deviations* 1 2 1 - 2 1 - - 4 2 4 - 2 19
% total deviations^ 1 2 1 - 2 1 - - 9 2 8 - 2 28
No. of tests 423 358 388 426 372 396 388 211 293 388 386 218 301 4548
2009 111 % critical deviations* 2.4 0.3 2.1 0.2 1.1 2.5 0.5 3.8 5.8 2.3 2.8 1.8 0.3 1.9
% total deviations^ 3.8 0.3 4.6 0.9 1.1 3.5 1.5 3.8 18.1 3.6 7.5 1.8 0.6 3.8
∞
^S→R & R→S & S↔I or I↔R (I, intermediate)
-, not determined
38
Table 16. Region-based categorization of EQAS participating laboratories’ performance of antimicrobial susceptibility tests for Shigella strains in 2009
Region No. of % correct % minor % major deviations % very major % critical % total Countries participating in the 2009
labs test result deviations (S→R)^ deviations deviations deviations iteration
(S↔I or I↔R)^ (R→ S)^ (R→ S & S → R)^ (S→R & R→S &
S↔I or I↔R)^
Algeria, Cameroon, Central African
Republic, Democratic Republic of Congo,
Africa 17 93.3 2.4 3.5 0.8 4.3 6.8 Ethiopia, Gambia, Ivory Coast, Kenya,
Madagascar, Mauritius, Nigeria, South
Africa, Sudan, Tunisia
Central Asia
& Middle 5 94.8 0.9 3.0 1.3 4.4 5.2 Iran, Israel, Jordan, Oman, Yemen
East
Barbados, Jamaica, Suriname, Trinidad and
Caribbean 4 95.6 1.5 0.7 2.2 2.9 4.4
Tobago
China 12 96.3 2.2 1.0 0.5 1.5 3.7 China
Albania, Belgium, Bosnia and Herzegovina,

Denmark, Finland, Greece, Ireland, Italy,
Europe 22 98.1 1.1 0.7 0.1 0.8 1.9 Lithuania, Luxembourg, Republic of
Moldova, Poland, Serbia, Slovak Republic,
Slovenia, United Kingdom
North
6 100.0 0.0 0.0 0.0 0.0 0.0 United States of America
America
Oceanic 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -
Russia 6 95.5 1.6 1.6 1.3 2.9 4.6 Belarus, Georgia, Russia
Argentina, Bolivia, Brazil, Chile, Colombia,
South Costa Rica, Cuba, Ecuador, Guatemale,
20 98.3 1.1 0.4 0.3 0.7 1.7
America Honduras, Mexico, Nicaragua, Panama,
Paraguay, Peru, Uruguay, Venezuela
Cambodia, India, Japan, Lao, Malaysia,
Southeast
18 94.1 3.9 0.3 1.7 2.0 5.9 Philippines, South Korea, Sri Lanka,
Asia
Taiwan, Thailand, Vietnam
39
Table 17. EQAS participating laboratories’ performance of Campylobacter strains identification
EQAS No. of Correct species Strain no. No. of % correct Deviating results (*)
iteration labs results identification
submitted
C. coli (9)
97 C. jejuni #1 92 87%
C. lari (3)
2003
C. jejuni (7)
97 C. coli #2 92 83% C. lari (4)
C. upsaliensis (4)
C. coli (11)
109 C. lari #1 95 80%
C. jejuni (8)
2004
C. coli (8)
109 C. jejuni #2 107 87% C. lari (4)
C. upsaliensis (2)
C. lari (3)
99 C. jejuni #1 86 90% C. coli (3)
C. upsaliensis (3)
2006
C. lari (19)
99 C. coli #2 94 66% C. jejuni (11)
C. upsaliensis (2)
C. jejuni (10)
142 C. lari #1 95 72% C. coli (9)
C. upsaliensis (7)
2007
C. lari (3)
142 C. coli #2 99 74% C. jejuni (20)
C. upsaliensis (2)
C. coli (14)
154 C. lari #1 105 63% C. jejuni (18)
C. upsaliensis (7)
2008
C. coli (10)
154 C. lari #2 105 60% C. jejuni (19)
C. upsaliensis (13)
C. upsaliensis (10)
131 C. coli #1 87 77% C. jejuni (9)
C. lari (1)
2009
C. upsaliensis (3)
131 C. jejuni #2 87 95%
C. lari (1)
*number of participants reporting the specified deviating result
40
Table 18. Region-based categorization of EQAS 2009 participating laboratories’ performance of Campylobacter strains identification
% strains
No. of No. of strains
Region correctly Countries participating in the 2009 iteration
labs identified
identified
Africa 8 13 53.8 Algeria, Cameron, Democratic Republic of Congo, Ethiopia, Tunisia
Asia & Middle East 3 5 40 Egypt, Israel, Oman
Caribbean 2 4 100 Barbados, Trinidad and Tobago
China 12 24 91.7 China
Cyprus, Czech Republic, Denmark, Finland, Germany, Greece, Hungary, Italy, Lithuania,
Europe 28 53 88.7 Luxembourg, Republic of Moldova, Poland, Serbia, Slovak Republic, Slovenia, Turkey
North America 10 19 89.5 Canada, United States of America
Oceania 2 4 100 Australia, New Zealand
Russia 2 4 100 Belarus, Georgia
Argentina, Brazil, Chile, Colombia, Costa Rica, Cuba, Guatemale, Paraguay, Perù, Uruguay,
South America 14 26 88.5 Venezuela
Southeast Asia 10 20 90 Cambodia, Japan, Philippines, South Korea, Sri Lanka, Taiwan, Thailand, Vietnam
41
Table 19. EQAS participants’ performance of Campylobacter strains antimicrobial susceptibility
testing
EQAS No. of % correct % major % very major % critical

iteration labs test results deviations deviations deviations
(S → R)^ (R → S)^ (R → S & S → R)^
2009 25 91.4 4.5 4.1 8.6

^S, susceptible; R, resistant
Table 20. Antimicrobial susceptibility test results (number of R/S) for the EQAS 2009
Campylobacter strains*
Antimicrobial^
Strain
CHL CIP ERY GEN NAL STR TET
WHO
1/17 0/23 21/2 0/21 0/20 3/13 2/20
C-9.1
WHO
2/15 19/2 2/19 0/20 16/3 1/15 19/2
C-9.2
^For antimicrobial abbreviations, see List of Abbreviations page 1

*In bold: expected interpretation. R, resistant; S, susceptible
Table 21. EQAS participants’ performance of Campylobacter antimicrobial susceptibility testing

categorized by antimicrobial
EQAS No. of Lab Antimicrobial

iteration labs performance
CHL CIP ERY GEN NAL STR TET
No. of tests 37 46 46 43 41 34 45
2009 25
% critical deviations* 8.1 6.5 10.8 2.3 9.8 11.8 11.1
^For antimicrobial abbreviations, see List of Abbreviations page 1
42
Table 22. Region-based categorization of EQAS 2009 participants’ performance of antimicrobial susceptibility testing of Campylobacter strains
Region No. of % correct % major % very major % critical Countries participating in the 2009 iteration
labs test result deviations deviations deviations
(S → R)^ (S → R)^ (R→S & S→R)^
Africa 2 50.0 21.4 28.6 50.0 Algeria, Tunisia
Central Asia & Middle East 0 - - - - -
Caribbean 0 - - - - -
China 2 95.2 4.8 0.0 4.8 China
Europe 9 98.3 1.7 0.0 1.7 Denmark, Greece, Italy, Luxembourg, Poland, Slovenia
North America 2 100.0 0.0 0.0 0.0 United States of America.
Oceania 0 - - - - -
Russia 0 - - - - -
South America 5 93.2 6.8 0.0 6.8 Argentina, Brazil, Costa Rica, Paraguay
Southeast Asia 4 71.4 0.0 28.6 28.6 Thailand, Phippines, Sri Lanka, Korea
^S, susceptible; R, resistant
43
Table 23. EQAS 2009 participants’ performance of antimicrobial susceptibility testing of Campylobacter
jejuni ATCC 33560
Incubation Labs’ Antimicrobial3

Method used
conditions performance1, 2
CHL CIP ERY GEN NAL TET
No.1 6 9 10 9 7 9
Microdilution 42°C / 24h
% 2
83.3 66.7 80 88.9 100 88.9
No. 1
5 5 5 5 5 5
Microdilution 36-37°C / 48h
% 2
80 80 80 80 80 80
EQAS No.1 0 5 5 6 0 0
2009 Agardilution 42°C / 24h
(N=24 % 2
- 100 40 66.7 - -
No. 1
0 2 2 2 0 0
Agardilution 36-37°C / 48h
% 2
- 100 100 100 - -
No. 1
11 21 22 22 12 14
Overall Overall
% 2
81.8 81 72.7 75 91.7 85.7
1
No., number of labs performing the analysis
2
%, percentage of labs reporting correct results
3
-, not determined
Table 24. EQAS participating laboratories’ performance of unknown strain identification

EQAS Strain ID No. of Pecentage (%) of labs
iteration participating labs performing correct
identification
2003 E. coli O157 115 99
94 (Shigella)
2004 Shigella flexneri 121
74 (S. flexneri)
93 (Yersinia)
2006 Yersinia enterocolitica O3 134 89 (Y. enterocolitica)
66 (Y. enterocolitica O3)
2007 Vibrio parahaemolyticus 86 83
2008 Enterobacter sakasakii 128 92
2009 Vibrio mimicus 56 48
44
Appendixes (1-4b)
45
APPENDIX 1
WHO Global Salm-Surv Electronic Discussion Group

Subject: Signing up for EQAS 2009
Greetings WHO Global Foodborne Infections Network (WHO GFN) Members:

WHO GFN strives to increase the quality of laboratory-based surveillance of Salmonella and other foodborne
pathogens by encouraging national or regional reference laboratories that have attended WHO GFN training
courses to participate in the External Quality Assurance System (EQAS). The 2008 EQAS cycle has closed,
and we are pleased to announce the launch of the 2009 EQAS cycle.
WHY PARTICIPATE IN EQAS?

EQAS provides the opportunity for proficiency testing. Proficiency testing is considered an important tool for
the production of reliable laboratory results of consistently good quality.
WHAT IS OFFERED IN EQAS?

This year’s WHO EQAS offers
− serogrouping, serotyping and antimicrobial susceptibility testing of eight Salmonella isolates;
− serotyping and antimicrobial susceptibility testing of four Shigella isolates;
− species identification and antimicrobial susceptibility testing of two Campylobacter isolates;
− identification of one unknown bacterial sample.
WHO SHOULD PARTICIPATE IN EQAS 2009?

All national or regional reference laboratories that are performing work on Salmonella, Shigella and/or
Campylobacter and are interested in participating in a quality assurance program are invited to participate.
We expect that all national or regional reference laboratories that have attended WHO GFN Training
Courses will participate in EQAS.
The WHO GFN Regional Centers, in cooperation with the EQAS Coordinator, will evaluate the list of
participants that wish to enroll in EQAS 2009. Laboratories which signed up and received strains in year
2008, but did not submit any data, should explain the reason for this in order to participate in 2009.
COST FOR PARTICIPATING IN EQAS

There is no charge for participating in EQAS 2009; however, laboratories which are capable of paying for
shipping the parcel should intend to do so. If your country has an agreement with FedEx, regarding importing
Biological Substance Category B (UN3373) please forward your FedEx import account number in the sign-
up form, or alternatively to the EQAS Coordinator (contact information below),. Having this information before
sending out the isolates saves time and resources. Participating laboratories are responsible for paying any
expenses related to getting the parcel through customs, additional taxes or customs fees.
SIGNING UP FOR THE EQAS 2009

This link will take you to a sign up webpage: http://thor.dfvf.dk/signup
You will be asked to fill in the following information:
− Name of institute, department, laboratory and contact person
− Complete mailing address for shipping (no post-office box number)
− Telephone, fax, e-mail
− FedEx import account number (if such one is available)
− Approximate number of Salmonella isolates annually serogrouped/serotyped
− Approximate number of Salmonella isolates annually tested for antimicrobial susceptibility
− Level of participation in EQAS 2009
− Level of reference function in your country
If you experience any problems enrolling electronically, please try again a few days later. If you are
still unsuccessful after attempting to enroll, please contact the EQAS Coordinator, Susanne
Karlsmose, by e-mail (suska@food.dtu.dk) or fax (+45 7234 6001).
SHIPPING AND TIMELINE TO RECEIVE ISOLATES AND PROTOCOLS

Due to the increased number of participants in EQAS, a number of different institutions will ship the bacterial
isolates. You will be informed of the institution which will ship your parcel. In order to minimize the delay in
APPENDIX 1
shipping the isolates to your laboratory, please provide the coordinator with a valid import permission.
Please apply for a permit to receive the following (according to your level of participation): “Biological
Substance Category B”: eight Salmonella strains, four Shigella strains, two Campylobacter, one
Campylobacter reference strain (for participants performing antimicrobial susceptibility testing on
Campylobacter), one E .coli reference strain (for new participants performing antimicrobial susceptibility
testing on Salmonella and/or Shigella) and an unknown sample (enteric bacteria) between August and
September 2009.
The isolates will be shipped between August and September 2009. The protocol as well as additional
information needed for EQAS will be made available for download from the website.
http://www.antimicrobialresistance.dk/233-169-215-eqas.htm.
TIMELINE FOR RESULTS TO BE TURNED INTO THE NATIONAL FOOD INSTITUTE

Results must be returned to the National Food Institute (DTU Food) by 31st of December 2009 via the
password protected website. Immediately upon receiving the results, an evaluation report will be generated.
Full anonymity is ensured; only DTU Food and the WHO Global Salm-Surv Regional Centre in your region
will be given access to your results.
Deadline for signing up to participate in this EQAS: April 30th, 2009

********************************************************************************
Posted by Susanne Karlsmose, suska@food.dtu.dk, WHO Global Foodborne Infections Network EQAS
Coordinator, DTU Food, The National Food Institute, Denmark.
APPENDIX 2
Salmonella Ampicillin Cefotaxime CTX/CL : CTX Ceftazidime CAZ/CL : CAZ Ceftriaxone Chloramphenicol Ciprofloxacin Gentamicin Nalidixic acid Streptomycin Sulfiz. Tetracycline Trimethoprim TriSul
AMP CTX CAZ CRO CHL CIP GEN NAL STR SMX TET TMP SXT
4 0.25 <0,25/0,125 1.0 <0,25/0,125 0.25 8 0.030 >16 4 64 >1024 4 <=1 0.125
WHO S-9.1 S. Enteritidis 9,12:g,m:-
SUSC SUSC non-ESBL SUSC non-ESBL SUSC SUSC SUSC RESIST SUSC RESIST RESIST SUSC SUSC SUSC
<=1 <=0.12 <0,25/0,125 0.25 <0,25/0,125 0.064 4 <=0.015 <=0.5 2 <=8 32 <=2 <=1 0.064
WHO S-9.2 S. Brandenburg 4,12:l,v:e,nz15
SUSC SUSC non-ESBL SUSC non-ESBL SUSC SUSC SUSC SUSC SUSC SUSC SUSC SUSC SUSC SUSC
<=1 <=0.12 <0,25/0,125 0.5 <0,25/0,125 0.125 8 <=0.015 1 4 <=8 <=16 <=2 <=1 0.064
WHO S-9.3 S. Muenster 3,10:e,h,1,5
<=1 <=0.12 <0,25/0,125 0.25 <0,25/0,125 0.064 4 0.030 <=0.5 2 <=8 <=16 <=2 <=1 0.064
WHO S-9.4 S. Bredeney 1,4,12:l,v,1,7
<=1 <=0.12 <0,25/0,125 0.25 <0,25/0,125 0.064 8 0.030 0.5 2 <=8 32 <=2 <=1 0.064
WHO S-9.5 S. Sandiego 4,5,12:e,h:e,n,z15
<=1 <=0.12 <0,25/0,125 0.25 <0,25/0,125 0.064 8 <=0.015 0.5 2 128 >1024 >32 >32 >32
WHO S-9.6 S. Worthington 1,13,23:z:l,w
SUSC SUSC non-ESBL SUSC non-ESBL SUSC SUSC SUSC SUSC SUSC RESIST RESIST RESIST RESIST RESIST
>32 <=0.12 <0,25/0,125 0.5 <0,25/0,125 0.125 >64 0.25 <=0.5 >64 <=8 >1024 >32 >32 >32
WHO S-9.7 S. Albany 8,20:z4,z24:-
RESIST SUSC non-ESBL SUSC non-ESBL SUSC RESIST RESIST SUSC RESIST SUSC RESIST RESIST RESIST RESIST
<=1 <=0.12 <0,25/0,125 0.25 <0,25/0,125 0.032 >64 <=0.015 <=0.5 4 <=8 >1024 >32 >32 >32
WHO S-9.8 S. Stanley 4,5,12:d:1,2
SUSC SUSC non-ESBL SUSC non-ESBL SUSC RESIST SUSC SUSC SUSC SUSC RESIST RESIST RESIST RESIST
Shigella Ampicillin Cefotaxime CTX/CL : CTX Ceftazidime CAZ/CL : CAZ Ceftriaxone Chloramphenicol Ciprofloxacin Gentamicin Nalidixic acid Streptomycin Sulfiz. Tetracycline Trimethoprim TriSul
AMP CTX CAZ CRO CHL CIP GEN NAL STR SMX TET TMP SXT
>32 <=0.12 <0,25/0,125 0.125 <0,25/0,125 0.016 <=2 <=0.015 <=0.5 1 >128 >1024 >32 >32 >32
WHO SH-9.1 S. sonnei
RESIST SUSC non-ESBL SUSC non-ESBL SUSC SUSC SUSC SUSC SUSC RESIST RESIST RESIST RESIST RESIST
4 <=0.12 <0,25/0,125 0.125 <0,25/0,125 0.032 <=2 <=0.015 <=0.5 1 <=8 <=16 <=2 <=1 0.064
WHO SH-9.2 S. flexneri type 6
>32 <=0.12 <0,25/0,125 0.125 <0,25/0,125 0.064 64 <=0.015 <=0.5 1 >128 >1024 >32 >32 >32
WHO SH-9.3 S. flexneri type 2a
RESIST SUSC non-ESBL SUSC non-ESBL SUSC RESIST SUSC SUSC SUSC RESIST RESIST RESIST RESIST RESIST
>32 <=0.12 <0,25/0,125 0.064 <0,25/0,125 0.032 <=2 <=0.015 <=0.5 1 64 >1024 32 <=1 0.064
WHO SH-9.4 S. boydii type 2
RESIST SUSC non-ESBL SUSC non-ESBL SUSC SUSC SUSC SUSC SUSC RESIST RESIST RESIST SUSC SUSC
Campylobacter Chloramph. Ciproflox. Erythromycin Gentamicin Nalidixic acid Streptom. Tetracycline

CHL CIP ERY GEN NAL STR TETRA
= 4 0.25 >32 0.5 = 8 <=1 = 2
WHO C-9.1 C. coli
SUSC SUSC RESIST SUSC SUSC SUSC SUSC
= 4 >4 = 2 0.5 >64 <=1 >16
WHO C-9.2 C. jejuni
SUSC RESIST SUSC SUSC RESIST SUSC RESIST
WHO B-9.1 Vibrio mimicus

WHO Collaborating Centre APPENDIX 3
External Quality Assurance System (EQAS) 2009
PROTOCOL for
- serotyping and susceptibility testing of Salmonella
- serotyping and susceptibility testing of Shigella
- identification and susceptibility testing of Campylobacter
- identification of an unknown enteric pathogen
1 INTRODUCTION ........................................................................................................................ 1
2 OBJECTIVES .............................................................................................................................. 2
3 OUTLINE OF THE EQAS 2009 ................................................................................................ 2
3.1 Shipping, receipt and storage of strains .........................................................................2
3.2 Serotyping of Salmonella .................................................................................................3
3.3 Susceptibility testing of Salmonella, Shigella and E. coli ATCC 25922 ......................3
3.4 Handling the Campylobacter strains ...............................................................................4
3.5 Identification of Campylobacter ......................................................................................5
3.6 Susceptibility testing of Campylobacter and C. jejuni ATCC 33560 ............................5
3.7 Identification and of the unknown enteric pathogen.....................................................6
4 REPORTING OF RESULTS AND EVALUATION ................................................................ 6
5 HOW TO ENTER RESULTS IN THE INTERACTIVE DATABASE .................................. 7
1 INTRODUCTION
In 2000, the Global Foodborne Infections Network (formely known as WHO Global Salm-Surv)
launched an External Quality Assurance System (EQAS). The EQAS is organized by the National
Food Institute, Technical University of Denmark (DTU Food), in collaboration with partners and
Regional Sites in WHO GFN.
Various aspects of the proficiency test scheme may from time to time be subcontracted. When
subcontracting occurs it is placed with a competent subcontractor and the National Food Institute is
responsible to the scheme participants for the subcontractor’s work.
The WHO EQAS 2009 includes
- serotyping and susceptibility testing of eight Salmonella strains,
- serotyping and susceptibility testing of four Shigella strains,
- susceptibility testing of the E. coli reference strain for quality control (ATCC 25922 (CCM
3954)),
Page 1 of 8
DFVF- M00-06-001/21.08.2009
- identification and susceptibility testing of two thermophilic Campylobacter isolates

- susceptibility testing of the C. jejuni reference strain for quality control (ATCC 33560
(CCM 6214)),
- and identification of one ‘unknown’ bacterial isolate.
All participants will receive the strains relevant to their laboratory according to the sign-up
information.
For new participants of the EQAS who have not already received the mentioned reference strains,
these are included in the parcel. The reference strains will not be included in the years to come. The
reference strains are original CERTIFIED cultures and are free of charge and should be used for
future internal quality control for susceptibility testing in your laboratory. Please take proper care of
the strains. Handle and maintain them as suggested in the manual ‘Subculture and Maintenance of
QC Strains’ available on the WHO CC website (see www.antimicrobialresistance.dk).
2 OBJECTIVES
The main objective of this EQAS is to support laboratories to assess and if necessary improve the
quality of serotyping and susceptibility testing of enteric human pathogens, especially Salmonella.
Furthermore, to assess and improve the comparability of surveillance data on Salmonella serotypes
and antimicrobial susceptibility reported by different laboratories. The laboratory work for this
EQAS should be done by the methods routinely used in your laboratory.
3 OUTLINE OF THE EQAS 2009
3.1 Shipping, receipt and storage of strains
In August/September 2009 around 190 laboratories from all parts of the world will receive a parcel
containing eight Salmonella strains, four Shigella, two Campylobacter strains and one ‘unknown’
bacterial isolate (according to information when signing up). An E. coli reference strain and a C.
jejuni reference strain will be included for participants who have signed up to perform antimicrobial
susceptibility testing (AST) and who have not previously received these. All strains are non-toxin
producing human pathogens Class II. There might be ESBL-producing strains among the selected
material.
Please confirm receiving the parcel by the confirmation form enclosed in the shipment.
The reference strains and the Campylobacter strains are shipped lyophilised, whereas the
Salmonella and Shigella strains, as well as the ‘unknown’ isolate are stab cultures. On arrival, the
stab cultures must be subcultured, and all cultures should be kept refrigerated until testing. A
suggested procedure for reconstitution of lyophilized strains is presented below.
Page 2 of 8
DFVF- M00-06-001/21.08.2009
3.2 Serotyping of Salmonella
The eight Salmonella strains should be serotyped by the method routinely used in the laboratory. If
you do not have all the antisera please go as far as you can, and please report the serogroup, since
also serogrouping results will be evaluated. When reporting serogroups, please use terms according
to Kaufman-White (Popoff and Le Minor, 2001. 8th ed. Popoff, M.U., Le Minor, L., 2001.
Antigenic formulas of the Salmonella serovars. WHO Collaborating Centre for Reference and
Research on Salmonella).
When uploading the data, please fill in the information on the brand of antisera used in the typing.
3.3 Susceptibility testing of Salmonella, Shigella and E. coli ATCC 25922
The Salmonella and Shigella strains as well as the E. coli reference strain should be susceptibility
tested towards as many as possible of the antimicrobials mentioned in the test form. Please use the
methods routinely used in the laboratory.
For reconstitution of the E. coli reference strain: Please see the document ‘Instructions for opening
and reviving lyophilised cultures’ on the WHO CC website (see www.antimicrobialresistance.dk).
Testing of gentamicin and streptomycin may be of value for monitoring. Please, do not take into
account in this study, that the CLSI guidelines state that for aminoglycosides Salmonella and
Shigella should not be reported as susceptible.
Antimicrobials Reference value, MIC (g/mL)

Sensitive Intermediate Resistant
Ampicillin, AMP* 8 16 32
Cefotaxime, CTX** 0.5 - >0.5
Ceftazidime, CAZ** 2 - >2
Ceftriaxone, CRO*** 0.25 - >0.25
Chloramphenicol, CHL* 8 16 32
Ciprofloxacin, CIP** <0.125 - 0.125
Gentamicin, GEN* 4 8 16
Nalidixic acid, NAL* 16 - 32
Streptomycin, STR*** 8 16 32
Sulfonamides, SMX* 256 - 512
Tetracycline, TET* 4 8 16
Trimethoprim, TMP* 8 - 16
Trimethoprim + sulfamethoxazole, TMP+SMX, SXT* 2/38 - 4/76
*CLSI **EUCAST (epidemiological cut off values) ***DTU Food
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DFVF- M00-06-001/21.08.2009
In this EQAS the breakpoints used as a key to interpreting MIC results are a mixture of reference
values from CLSI, EUCAST and DTU Food (see list above). This allows three categories of
characterisation – resistant, intermediate or sensitive. Interpretations in concordance with the
expected value will be categorised as ‘correct’, whereas deviations from the expected interpretation
are categorizes as ‘minor’ (I  S or I  R), ‘major’ (S interpreted as R) or ‘very major’ (R
interpreted as S).
As to the breakpoints that you routinely use in your laboratories to determine the susceptibility
category we ask you to fill in these breakpoints in the database (or in the test form).
For ciprofloxacin, please note that a low breakpoint has been used to determine resistance category.
Considering the expected results of this EQAS, microorganisms are considered resistant to
ciprofloxacin when showing reduced susceptibility to this antimicrobial.
ESBL production
It is optional to continue with the following tests regarding ESBL production:
All strains categorized reduced susceptible against cefotaxime (CTX), ceftazidime (CAZ) or
ceftriaxone (CRO) could be confirmed by confirmatory tests for ESBL production.
The confirmatory tests require testing with a pure antimicrobial (CTX and CAZ) vs. a test with the
same antimicrobial combined with a -lactamase inhibitor (clavulanic acid). Synergy is defined as a
3 dilution steps difference between the two compounds in at least one of the two cases (MIC ratio 
8, E-test 3 dilution steps) or an increase in zone diameter  5 mm (CLSI M100 Table 2A;
enterobacteriaceae). If the test shows signs of synergy it is an indication of the presence of ESBL.
Concerning cefotaxime (CTX), ceftazidime (CAZ) and/or ceftriaxone (CRO) used when detecting
ESBL-producing strains in the EQAS: If a microorganism is resistant to one or two of these drugs,
it should be regarded resistant to all three.
3.4 Handling the Campylobacter strains
Freeze-dried cultures are supplied in vacuum-sealed ampoules. Care should be taken in opening the
ampoule. All instructions given below should be followed closely to ensure the safety of the person
who opens the ampoule and to prevent contamination of the culture.
a. Check the number of the culture written on the label.

b. Make a file cut on the ampoule just above the shoulder of the ampoule.
c. Disinfect the ampoule with alcohol-dampened gauze or alcohol-dampened cotton wool.
d. Crack the glass using sterile gauze or cotton to protect your fingers.
e. Add to the dried suspension about 0.5 ml appropriate broth or a sterile 0.9% NaCl solution
using a pipette. Mix carefully to avoid creating aerosols.
Page 4 of 8
DFVF- M00-06-001/21.08.2009
f. Inoculate the suspension on a suitable agar plate with a 10µl loop or a cotton swab.
g. Transfer the rest of the content in the ampoule to a test tube containing 5-6 ml of a suitable
liquid media.
h. Incubate the agar plate and liquid media at a temperature of 42°C at microaerobic conditions
for 24-48 hours.
i. Inoculate a second agar plate from the liquid media with a 10µl loop or a cotton swab if the
initial plate had inadequate growth.
j. Select a pure culture with vigorous growth from the agar plate for further work.
Please note that:
 Cultures may need at least one sub-culturing before they can be optimally used
 Unopened ampoules should be kept in a dark and cool place!
For reconstitution of the C. jejuni reference strain: Please see the document ‘Instructions for
opening and reviving lyophilised cultures’ on the WHO CC website (see
www.antimicrobialresistance.dk).
3.5 Identification of Campylobacter
The two thermophilic Campylobacter isolates should be identified to species level.
3.6 Susceptibility testing of Campylobacter and C. jejuni ATCC 33560
The Campylobacter test strains as well as the C. jejuni reference strain should be susceptibility
tested towards as many as possible of the antimicrobials mentioned in the test form. It should be
noted that for AST of Campylobacter only MIC methods are recommendable, i.e. broth or agar
dilution methods. Neither the use of disk diffusion nor E-test is recommendable for AST of
Campylobacter.
In this EQAS the breakpoints used as a key to interpreting MIC results for Campylobacter are
epidemiological cut off values. The reference values used are from EUCAST (www.eucast.org; see
list below). This allows only two categories of characterisation – resistant or sensitive.
Interpretations in concordance with the expected value will be categorised as ‘correct’, whereas
deviations from the expected interpretation are categorizes as ‘incorrect’.
As to the breakpoints that you routinely use in your laboratories to determine the susceptibility
category we ask you to fill in these breakpoints in the database (or in the test form).
Page 5 of 8
DFVF- M00-06-001/21.08.2009
Note that the interpretation requires knowledge about the species. If you do no identify
Campylobacter but perform AST on Campylobacter, you may contact the EQAS Coordinator to
obtain information regarding the identity of the Campylobacter test strains.
MIC (g/mL) MIC (g/mL)
Antimicrobials for Campylobacter
R is > R is >
C. jejuni C. coli
Chloramphenicol 16 16
Ciprofloxacin 1 1
Erythromycin 4 16
Gentamicin 1 2
Nalicixic acid 16 32
Streptomycin 2 4
Tetracycline 2 2
The sub-cultured Campylobacter should be used for the MIC-testing after incubation at 36ºC for 48
hours or 42ºC for 24 hours; possibly two subcultures are needed to ensure good growth before
testing.
3.7 Identification and of the unknown enteric pathogen
The ‘unknown’ isolate should be identified to species level and further typed if relevant.
4 REPORTING OF RESULTS AND EVALUATION
Fill in your results in the enclosed test form and enter your results into the interactive web database.
Please read the detailed description below before entering your results. When you enter the results
via the web, you will be guided through all steps on the screen and you will immediately be able to
view and print an evaluation report of your results. Please submit results by latest December 31st,
2009. If you do not have access to the Internet or if you experience difficulties entering the data,
please return results by fax or mail to the National Food Institute.
All results will summarized in a report which will be made available to all participants. Individual
results will be anonymous and will only be passed on to the official GFN Regional Centre in your
region.
We are looking forward to receiving your results.
If you have any questions or concerns, please do not hesitate to contact the EQAS
Coordinator:
Ms. Susanne Karlsmose
National Food Institute, Technical University of Denmark

Page 6 of 8
DFVF- M00-06-001/21.08.2009
27 Bülowsvej, DK-1790 Copenhagen V - DENMARK
Tel: +45 3588 6601, Fax: +45 3588 6001
E-mail: suska@food.dtu.dk
It is possible to communicate with the EQAS organisers in other languages than English. However,
this is not a direct contact with the EQAS organisers since translation of the message is required.
The following languages may be used: Russian, Chinese, French, Spanish or Portuguese.
5 HOW TO ENTER RESULTS IN THE INTERACTIVE DATABASE
Please read this passage before entering the web page. Before you go ahead, you need your test
form by your side together with your breakpoint values.
In general you navigate in the database with the Tab-key and mouse, and at any time a click on the
WHO logo takes you back to the main menu.
1) Enter the WHO CC website (from http://www.antimicrobialresistance.dk), then

a. Click on ‘EQAS’
b. Click on the link for the interactive database
c. Write your username and password in low letters and click on ‘Login’.
In the letter following your parcel you can find your username and password.
Your username and password will be the same in future trials.
2) Click on ‘Materials and methods’

a. Fill in the brand of antisera (very important as we would like to compare results with the
brand of antisera
b. Fill in the method used for susceptibility testing
c. Enter the brand of accessories, e.g. Oxoid
d. Fill in whether your institute serves as a national reference laboratory
e. Click on ‘Save and go to next page’ – REMEMBER TO SAVE EACH PAGE LIKE THIS!
3) In the data entry page ‘Routinely used breakpoints’

a. Fill in the breakpoints that you routinely use in your laboratory to determine the
susceptibility category. Remember to use the operator keys in order to show – equal to,
less than, less or equal to, greater than or greater than or equal to.
4) In the data entry pages ‘Salmonella strains 1-8’, you

a. SELECT the serogroup (O-group) from the pop-up list, DO NOT WRITE – Wait a few
seconds – the page will automatically reload, so that the pop-up in the field “Serotype”
only contains serotypes belonging to the chosen serogroup.
Page 7 of 8
DFVF- M00-06-001/21.08.2009
b. SELECT the serotype from the pop-up list – DO NOT WRITE – wait a few seconds and
you can enter the antigenic formula (e.g. 1,4,5,12:i:1,2)
c. Enter the zonediameters in mm or MIC values in µg/ml. Remember to use the operator
keys to show e.g. equal to, etc.
d. Enter the interpretation as R, I or S
e. If you have performed confirmatory tests for ESBL producing strains, please choose the
test result from the pick list.
f. Fill in comments if relevant e.g. which antisera you miss for complete serotyping
g. Click on ‘Save and go to next page’
If you have not performed these tests please leave the fields empty
5) In the data entry page ‘E. coli reference strain’:

a. Enter the zonediameters in mm or MIC values in µg/ml. Remember to use the operator
keys to show e.g. equal to, etc.
b. Click on ‘Save and go to next page’
6) In the page ‘Identification of Campylobacter and unknown sample’:

a. Choose the correct Campylobacter species from the pick list
b. Fill in the species and type of the unknown bacterial isolate, and fill in the method used
c. Click on ‘Save and go to next page’
If you have not performed these tests please leave the fields empty
7) The next page is a menu, from where you can review the input pages or approve your input and
finally see and print the evaluated results
a. Browse through the input pages and make corrections if necessary. Remember to click on
‘save and go to next page’ if you make any corrections.
b. Approve your input. Be sure that you have filled in all the results before approval, as .YOU
CAN ONLY APPROVE ONCE!. The approval blocks your data entry in the interactive
database, but allows you to see the evaluated results.
c. As soon as you have approved your input, an evaluation report will show.
8) When you have seen all pages in the report, you will find a new menu. You can choose ‘EQAS
2009 start page’, ‘Review evaluated results’ (a printer friendly version of the evaluation report is
also available) or ‘Go to Global Salm-Surv homepage’.
End of entering your data – thank you very much!
Page 8 of 8
DFVF- M00-06-001/21.08.2009
WHO Collaborating Centre APPENDIX 4a
External Quality Assurance System (EQAS)
SUBCULTURE AND MAINTENANCE OF

QUALITY CONTROL STRAINS
1.1 Purpose
Improper storage and repeated subculturing of bacteria can produce alterations in antimicrobial
susceptibility test results. The Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS)
has published a guideline for Quality Control (QC) stock culture maintenance to ensure consistent
antimicrobial susceptibility test results.
1.2 References
M100-S18, January 2008 (Performance Standards for Antimicrobial Susceptibility Testing)
M7-A7, January 2006 (Methods for Dilution Antimicrobial Susceptibility Test for Bacteria That
Grow Aerobically; Approved Standard)
1.3 Definition of Terms

Reference Culture: A reference culture is a microorganism preparation that is acquired from a
culture type collection.
Reference Stock Culture: A reference stock culture is a microorganism preparation that is derived
from a reference culture. Guidelines and standards outline how reference stock cultures must be
processed and stored.
Working Stock Cultures: A working stock culture is growth derived from a reference stock culture.
Guidelines and standards outline how working stock cultures must be processed and how often they
can be subcultured.
Subcultures (Passages): A subculture is simply the transfer of established microorganism growth on
media to fresh media. The subsequent growth on the fresh media constitutes a subculture or
passage. Growing a reference culture or reference stock culture from its preserved status (frozen or
lyophilized) is not a subculture. The preserved microorganism is not in a stage of established
growth until it is thawed or hydrated and grown for the first time
1.4 Important Considerations

 Do not use disc diffusion strains for MIC determination.
 Obtain QC strains from a reliable source such as ATCC
 CLSI requires that QC be performed either on the same day or weekly (only after 30 day QC
validation)
 Any changes in materials or procedure must be validated with QC before implemented
 For example: Agar and broth methods may give different QC ranges for drugs such as
glycopeptides, aminoglycosides and macrolides
Subculture and Maintenance of QC strains DFVF-M00-06-001/31.10.2008
Page 1 of 4
 Periodically perform colony counts to check the inoculum preparation procedure

 Ideally, test values should be in the middle of the acceptable range
 Graphing QC data points over time can help identify changes in data helpful for
troubleshooting problems
1.5 Storage of Reference Strains

Preparation of stock cultures
 Use a suitable stabilizer such as 50% fecal calf serum in broth, 10-15% glycerol in tryptic
soy broth, defibrinated sheep blood or skim milk to prepare multiple aliquots.
 Store at -20°C, -70°C or liquid nitrogen. (Alternatively, freeze dry.)
 Before using rejuvenated strains for QC, subculture to check for purity and viability.
Working cultures
 Set up on agar slants with appropriate medium, store at 4-8°C and subculture weekly.
 Replace the working strain with a stock culture at least monthly.
 If a change in the organisms inherent susceptibility occurs, obtain a fresh stock culture or a
new strain from a reference culture collection e.g. ATCC.
1.6 Frequency of Testing

Weekly vs. daily testing
Weekly testing is possible if the lab can demonstrate satisfactory performance with daily testing as
follows:
 Documentation showing reference strain results from 30 consecutive test days were within
the acceptable range.
 For each antimicrobial/organism combination, no more than 3 out of 30 MIC values may be
outside the acceptable range.
When the above are fulfilled, each quality control strain may be tested once a week and whenever
any reagent component is changed.
Corrective Actions
If an MIC is outside the range in weekly testing, corrective action is required as follows:
 Repeat the test if there is an obvious error e.g. wrong strain or incubation conditions used
 If there is no obvious error, return to daily control testing
The problem is considered resolved only after the reference strain is tested for 5 consecutive days
and each drug/organism result is within specification on each day.
If the problem cannot be resolved, continue daily testing until the errors are identified.
Repeat the 30 days validation before resuming weekly testing.
Page 2 of 4
DAILY MIC QC CHART
Reference: CLSI M7-A7, page 39
Page 3 of 4
WEEKLY MIC QC CHART
Page 4 of 4
WHO Collaborating Centre APPENDIX 4b
INSTRUCTIONS FOR OPENING AND REVIVING

LYOPHILISED CULTURES
Manual from Czech Collection of Microorganisms (CCM)

Masaryk University
Tvrdého 14
602 00 BRNO
Czech Republic
Lyophilised cultures are supplied in vacuum-sealed ampoules. Care should be taken in opening the
ampoule. All instructions given below should be followed closely to ensure the safety of the person
who opens the ampoule and to prevent contamination of the culture.
a. Check the number of the culture on the label inside the ampoule
b. Make a file cut on the ampoule near the middle of the plug
c. Disinfect the ampoule with alcohol-dampened gauze or alcohol-dampened cotton wool from
just below the plug to the pointed end
d. Apply a red-hot glass rod to the file cut to crack the glass and allow air to enter slowly into
the ampoule
e. Remove the pointed end of the ampoule into disinfectant
f. Add about 0.3 ml appropriate broth to the dried suspension using a sterile Pasteur pipette
and mix carefully to avoid creating aerosols. Transfer the contents to one or more suitable
solid and /or liquid media
g. Incubate the inoculated medium at appropriate conditions for several days
h. Autoclave or disinfect effectively the used Pasteur pipette, the plug and all the remains of
the original ampoule before discarding
Please note that:

 Cultures should be grown on media and under conditions as recommended in the CCM
catalogue
 Cultures may need at least one subculturing before they can be optimally used in experiments
 Unopened ampoules should be kept in a dark and cool place!
Instructions for Opening and Reviving Lyophilised Cultures DFVF-M00-06-001/31.10.2008

Page 1 of 1
Technical University of Denmark
Mørkhøj Bygade 19
DK - 2860 Søborg
T: 35 88 70 00
F: 35 88 70 01
www.food.dtu.dk
ISBN: 978-87-92158-88-8

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The External Quality Assurance System of the

WHO Global Foodborne Infections Network 2009

National Food Institute

THE EXTERNAL QUALITY ASSURANCE SYSTEM OF

DATE OF ISSUE: DECEMBER 2010

1. edition, December 2010

The report is available at

National Food Institute

List of Abbreviations ..................................................................................................................................... 1

2. Materials and Methods

3.1 Methods used by EQAS participants

3.2 Serogrouping and serotyping of Salmonella strains

In Table 3 , t he pe rformance of Salmonella serotyping is r eported on a region-based categorization of

3.3 Antimicrobial susceptibility testing (AST) of Salmonella strains

3.4 Serogrouping and serotyping of Shigella strains

3.5 Antimicrobial susceptibility testing (AST) of Shigella strains

3.6 Identification of Campylobacter strains

In Table 18, the p erformance of Campylobacter identification i s r eported a ccording t o geographical

No major di fficulties in a ssessing antimicrobial s usceptibility w ere e ncountered for an y of t he t ested

3.8 Identification of the unknown culture

4.2 Antimicrobial susceptibility testing (AST) of Salmonella strains

4.3 Serogrouping and serotyping of Shigella strains

4.4 Antimicrobial susceptibility testing (AST) of Shigella strains

4.6 Antimicrobial susceptibility testing (AST) of Campylobacter strains

4.7 Identification of the unknown culture

Figure 1. Countries participating* in the WHO EQAS 2009

EQAS Labs serotyping all

Number Participating laboratories

Number Participating laboratories

Carno (3), Nanga (3), Tanzania (2),

Corvallis (3), Altona (2), Tallahassee (2),

Typhimurium (5), Duisburg (3),

EQAS Labs serotyping

^For antimicrobial abbreviations: see List of Abbreviations page 1

EQAS No. of Antimicrobial∞

2004 11 85.7 7.2 5.2 1.9 7.1 14.3

8 89.9 4.5 4.0 1.6 5.6 10.1

2004 8 96.0 3.2 0.7 0.1 0.8 4.0

2004 11 97.1 2.5 0.3 0.1 0.4 2.9

2004 4 2.2 8.8 Belarus, Georgia,

2003 MIC & Disk

WHO S. flexneri 2a (1), 2b (1),

WHO S. flexneri 6 (1), 4a (1),

EQAS No. of Lab Antimicrobial

China 12 96.3 2.2 1.0 0.5 1.5 3.7 China

Albania, Belgium, Bosnia and Herzegovina,

Oceanic 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -

EQAS No. of % correct % major % very major % critical

2009 25 91.4 4.5 4.1 8.6

^For antimicrobial abbreviations, see List of Abbreviations page 1

Table 21. EQAS participants’ performance of Campylobacter antimicrobial susceptibility testing

EQAS No. of Lab Antimicrobial

Incubation Labs’ Antimicrobial3

Table 24. EQAS participating laboratories’ performance of unknown strain identification

2003 E. coli O157 115 99

2007 Vibrio parahaemolyticus 86 83

2008 Enterobacter sakasakii 128 92

2009 Vibrio mimicus 56 48

WHO Global Salm-Surv Electronic Discussion Group