update on n. gonorrhoeae susceptibility...

Update on N. gonorrhoeae susceptibility testing

Dr Michelle Cole, Public Health EnglandBSAC Antimicrobial Susceptibility Testing User DayLondon, 18 June 2019

Gonorrhoea - disease

• Neisseria gonorrhoeae - Gram negative diplococci

• Primarily a mucosal infection – genital symptoms include discharge / pain

on urination

• Urethral infection in men: 90% symptomatic

• Women: 50% asymptomatic

• Pelvic inflammatory disease

• Ectopic pregnancy

• Infertility

• Extra-genital infections; eye, throat and rectum. Usually asymptomatic

2 Update on N. gonorrhoeae susceptibility testing

Gonorrhoea is the 2nd most commonly

diagnosed bacterial STI in England

249%

From 2009 to 2018

Source: Public Health England, GUMCAD

•56,259 diagnoses in England, 2018

-

5

10

15

20

25

30

35

40

45

50P

erc

en

tag

e o

f is

ola

tes

Year

Ceftriaxone Azithromycin Cefixime Ciprofloxacin Penicillin Tetracycline Spectinomycin

Percentage of gonococcal isolates resistant to selected antimicrobials: England and Wales,

2000-2017

Update on N. gonorrhoeae susceptibility testing4 GRASP – Gonococcal antimicrobial surveillance programme

Ceftriaxone MIC distribution: 2008 to 2014

Neisseria gonorrhoeae antimicrobial resistance trends in England and Wales

0

10

20

30

40

50

60

70

80

90

0.004 0.008 0.015 0.03 0.06 0.125 0.25

Pe

rce

nta

ge

of

iso

late

s

MIC

2008 2009 2010 2011 2012 2013 2014

Isolates less sensitive


0

10

20

30

40

50

60

70

80

90

0.004 0.008 0.015 0.03 0.06 0.125 0.25

Pe

rce

nta

ge

of

iso

late

s

MIC

2008 2009 2010 2011 2012 2013 2014 2015


Ceftriaxone MIC distribution: 2008 to 2015Sentinel

surveillance


0

10

20

30

40

50

60

70

80

90

0.004 0.008 0.015 0.03 0.06 0.125 0.25

Pe

rce

nta

ge

of

iso

late

s

MIC

2008 2009 2010 2011 2012 2013 2014 2015 2016



Sentinel

surveillance


0

10

20

30

40

50

60

70

80

90

0.004 0.008 0.015 0.03 0.06 0.125 0.25

Pe

rce

nta

ge

of

iso

late

s

MIC

2008 2009 2010 2011 2012 2013 2014 2015 2016 2017



• Shift in the modal MIC from 0.008 mg/L in 2016 to 0.015 mg/L in 2017

Sentinel

surveillance

GC Super bugs

9 Antimicrobial resistant Neisseria gonorrhoeae: a global perspective

3 cases of XDR-NG detected in England, 2018

March (SE Asia)

September (Ibiza –British partner)

November (UK – partner had sex in Ibiza)

Update on N. gonorrhoeae susceptibility testing10

2018/19 = 2x Chinese community

Impermeable to antibiotic

Modification

of target

Efflux of

antibiotic

Enzymatic modification/

degradation of antibiotic

Adept at developing and disseminating mechanisms of resistance

to therapeutic agents


500mg ceftriaxone

(IM)

1g azithromycin

(orally)

The recommended first-line

treatment for GC was dual therapy

+

Source: GRASP 2018 report (data for 2017 from sentinel sexual health clinics in England and Wales)

Drift in ceftriaxone modal MIC

10% of gonococcal isolates resistant

to azithromycin


1g ceftriaxone (IM)

Usually administered empirically

The recommended first-line

treatment for GC is monotherapy

Source: BASHH national guideline for the management of infection with Neisseria gonorrhoeae (2019)


NG AST – guidanceEUCAST - harmonising the European clinical breakpoints using an evidence based approach

• pharmacokinetic/pharmacodynamics data

• clinical therapeutic efficacy evidence

EUCAST disc method (2009) – however, there is currently NO EUCAST disc diffusion method for

susceptibility testing Neisseria species.

• MIC determination is recommended using EUCAST clinical breakpoints.

2016-8 - Period of grace, where laboratories could keep with the BSAC disc method (IsoSensitest

agar + 5% horse blood).

• However EUCAST MIC breakpoints should be adhered to.

2018 - BSAC now recommends that MIC testing is performed with 2x control strains; ATCC 49226

and WHO X

56,000 cases/year, approximately 50% with culture = 28K AST/year

• Disc diffusion is a finically attractive option.

However, zone diameters breakpoints for interpretation of susceptibility poorly correlate MIC

breakpoints, especially those close to the breakpoints & problems with supporting growth on some

media.

Need to adhere to EUCAST MIC breakpoints, but no AST methodology for NG is described

Most laboratories use gradient strips for MIC testing, with manufacturer’s recommended media

(e.g. Biomerieux Etests; refer to CLSI guidelines = GC agar and 1% defined growth supplement).

The recommended media for use with gradient strips is sometimes not easily accessible

• Alternative media which support gonococcal growth are more easily available.

NG AST – the problem

Azithromycin MICs (mg/L)

PHE GC-VIT MH-F

Biomerieux Etest Liofilchem Etest Biomerieux Etest Liofilchem Etest

WHO K 0.25 1 2 4

WHO P 4 8 16 >256

WHO V >256 >256 >256 >256

H18-134 4 16 32 >256

H18-386 64 >256 >256 >256

An example…


The studyCurrently, recommended media for gradient strip susceptibility testing of

N. gonorrhoeae is poorly available. We aimed to study the efficacy of a variety of pre-

poured media available in the UK

1. Growth comparison of Mueller Hinton-fastidious agars

No MH-F agar was found to be appropriately support the growth of a range of

gonococcal isolates

2. Growth comparison on six commercially available pre-poured alternative media

6 media from three manufacturers:

• Oxoid: Chocolate vitox, GC vitox, GC non-selective with lysed blood

• Becton Dickinson: GC chocolate, Mueller Hinton Chocolate

• E&O Labs: Mueller Hinton Chocolate

5 control strains and 5 ‘poor growers’ assessed for viability

Specialist Antimicrobial Chemotherapy Unit (SACU) – sub-cultured from chocolate and 0.5 McFarland

inocula were prepared using Mueller Hinton Broth (MHB)

PHE - sub-cultured from GCVIT and 0.5 McFarland inocula were prepared using saline

RESULTS: Agar best at supporting growth: GC vitox (Oxoid), GC non-selective with lysed blood

(Oxoid) and GC chocolate (Becton Dickinson)


3. Comparison of ceftriaxone and azithromycin MICs on six commercially available

pre-poured media

3 control strains (WHO F, G, K) tested on same 6 media using ceftriaxone and azithromycin

gradient strips (Biomerieux Etests).

RESULTS: MICs for both azithromycin and ceftriaxone on all media tested were in general

comparable and within range.

MICs were comparable between laboratories, however were generally lower in SACU.

Primary culture plate (chocolate) most likely responsible.

Further MIC determination will be performed using inocula prepared from culture grown on

GCVIT media.


4. Comparison of ceftriaxone and azithromycin MICs on six commercially

available pre-poured media

Using 2 WHO control N. gonorrhoeae strains (WHO G, K) and 50 N. gonorrhoeae isolates submitted to

GRASP 2017-8

Same methods as previous however all sub-culture from GCVIT



Azithromycin Ceftriaxone

SADU/PHE SADU/PHE/GRASP SADU/PHE SADU/PHE/GRASP

Oxoid GC + vitox 100 86 98 82

Oxoid GC Non-sel agar w

lysed blood

98 74 100 62

BD GC Chocolate 96 92 100 88

BD Mueller Hinton

Chocolate agar

96 78 100 57

Oxoid Chocolate + vitox n/a* 92* n/a* 31*

E&O Mueller Hinton

Chocolate agar

n/a* 74* n/a* 75*

GRASP = agar dilution with DST agar, 5% horse blood and 1% vitox

*No data from PHE

Percentage essential agreement


Growth Ease of reading

Oxoid GC + vitox 291 232

BD GC Choc 279 205

E&O 269 199

BD MH Choc 260 200

E&O 269 199

Oxoid choc + vitox 239 195

Oxoid non sel 196 194

Next steps1. Consider eliminating some media

2. Evaluate isolates with a wider range of MICs (categorical agreement)

3. Include PHE GCVIT media

4. Establish MICs of ‘best’ media using agar dilution

5. Evaluate CRO, AZM, CIP gradient strips from different manufacturers on ‘best’ media

6. Evaluate discs on the media and try to establish a disc diffusion methodology ??

Funding required = to provide a robust reference method and media options for UK laboratories to

maintain high quality susceptibility testing for N. gonorrhoeae


To concludeSaline comparable diluent to MH-broth

Two current front runners, however more data is required: Oxoid GC + vitox and BD GC Chocolate

In the lab, ideally focus on ceftriaxone and azithromycin.

• Spectinomycin difficult to source

• Gentamicin no breakpoints

• Ciprofloxacin resistance too high (but perhaps requested 2nd/3rd line)

Perform validation using control strains – available from NCTC

Send any CRO-R and HL-AZR (MIC >256 mg/L) isolates to PHE for confirmation

Note: Azithromycin clinical breakpoints removed. Ecoff > 1mg/L


The study group & acknowledgements


Specialist Antimicrobial Chemotherapy unit (SACU): Mandy Wootton & Leanne

Davies

PHE NG Reference laboratory: Michelle Cole, Fazila Ismail, Helen Fifer and Neil

Woodford

BSAC: Carolyne Horner

EUCAST Development Laboratory (EDL): Gunnar Kahlmeter

National Reference Laboratory for Pathogenic Neisseria (NRLPN): Magnus Unemo

Any questions: [email protected]

mailto:[email protected]

update on n. gonorrhoeae susceptibility...

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