1. Determination of the antimicrobial susceptibility of Neisseria gonorrhoeae
Trevor Winstanley Rebecca Clarke
Department of Microbiology
Royal Hallamshire Hospital
Sheffield UK
29 July Freeman Hospital

2. Gonorrhoea 2nd most common bacterial STD Highest incidence
> 22,500 episodes diagnosed in GUM clinics in England & Wales (2001)
Highest incidence
Males /100,000
Females /100,000
VITEK 2 and AES have been evaluated in several countries. Study extended to a comparison with routine methodology.

3. Epidemiology
Concentrated within demographic and behavioural risk groups
High-levels of
re-infection
concurrent STDs
asymptomatic infection
Concentrated within demographic and behavioural risk groups Gay and bisexual men; black and ethnic minorities
High-levels of re-infection 33%
High-levels of concurrent STDs 31% at least one other STD (especially females)
High-levels of asymptomatic infection 42% females; 13% males

4. Public health concern Increasing incidence
Poor reproductive and sexual health outcomes
High prevalence of resistance
onward transmission
adverse clinical sequelae

5. Antimicrobial resistance
10 -12% of gonococcal isolates are resistant to some degree
inner cities
those acquiring infections abroad
gay and bisexual men

6. Plasmid-mediated PPNG TRNG PP/TRNG ß-lactamase Tetracycline Positive
< 16 mg/L
TRNG
Negative
 16 mg/L
PP/TRNG

7. Chromosomal PenR  1 mg/L TetR CMRNG ß-lactamase Penicillin
Tetracycline
PenR
Negative
 1 mg/L
< 2 mg/L
TetR
< 1 mg/L
2 - 8 mg/L
CMRNG

8. Chromosomal  0.125 < 1 mg/L  1 mg/L  128 mg/L  0.5 mg/L
Ciprofloxacin
Intermediate
 < 1 mg/L
Resistant
 1 mg/L
Spectinomycin
 128 mg/L
Ceftriaxone
Decreased
 0.5 mg/L
Azithromycin
Ciprofloxacin – single and double chromosomal mutations
Reduced – gyrA
High-level – gyrA and parC
Clinical failure at 0.06 – 0.25 with cipro 250 mg.

9. U.K. guidelines Easily treatable Penicillins Fluoroquinolones
95% cure from 1st line therapy
Penicillins
Fluoroquinolones
ciprofloxacin, ofloxacin
Cephalosporins
ceftriaxone, cefixime
(Doxycycline / tetracycline)
Easily treated with appropriate antimicrobials
1st line therapy should eradicate 95% of uncomplicated infection in the community

10. GRASP Gonococcal Resistance to Antimicrobials Surveillance Programme
DoH sentinel surveillance
PHLS Communicable Disease Surveillance Centre (CDSC)
PHLS Genitourinary Infections Reference Laboratory (GUIRL)
Department of Infectious Diseases & Microbiology at Imperial College
Sentinel surveillance in England & Wales established 2000 sponsored by Department of Health.
“Gonococcal Resistance to Antimicrobials Surveillance Programme” GRASP
Analysis and co-ordination
PHLS Communicable Disease Surveillance Centre (CDSC)
Testing
PHLS Genitourinary Infections Reference Laboratory (GUIRL), Bristol Department of Infectious Diseases & Microbiology at Imperial College
Now Colindale
Epidemiological data + consecutive isolates, 26 clinics, 3 months each summer isolates 2001
Reference MIC method.

11. GRASP 2001 (n = 2666) Penicillin Plasmid 3.0% Chromosome 5.1%
Tetracycline
4.6%
27.9%
Ciprofloxacin
Intermediate
2.6%
Resistant
3.1%
Azithromycin
0.3%
Spectinomycin ND
Ceftriaxone
Treatment failures with CIP R strains in Australia, UK, Canada and USA. Treatment also fails with CIP I strains.
CIP R: 10% Hong Kong and Japan, 62% Philippines.
Regional variation e.g. high resistance to CIP outside London.

12. Cure rates Uncomplicated genital gonorrhoea Recommended dosage
Susceptible > 95%
Intermediate %
Resistant < 90%
WHO; SRGA; NCCLS
Excellent correlation between susceptibility and clinical response

13. Media NCCLS SRGA BSAC ARMRL GRASP NCCLS GC agar base + 1% Isovitalex
SRGA –as above + 1% haemoglobin
BSAC – don’t want two different media
GRASP – makes sense that routine labs use the same medium as the reference centre

14. To validate the BSAC disc diffusion method for N.gonorrhoeae
Objectives
To validate the BSAC disc diffusion method for N.gonorrhoeae
to translate reference into routine
To confirm or refute tentative breakpoints
To extend the range of interpretive criteria

15. Methodology 222 distinct isolates from 5 geographical regions
5 WHO control strains
Disc diffusion tests, MICs
BSAC methodology
ß-lactamase
Nitrocefin
City Hospital Birmingham,
St Thomas’s
New Cross Wolverhampton
Stoke on Trent PHL
GC Reference Lab Bristol

16. Penicillin

17. Amoxycillin

18. Co-amoxyclav

19. Ciprofloxacin
Classic saw-tooth appearance of somebody doubling radii to make diameters.

20. Nalidixic acid

21. Tetracycline

22. Metzler & DeHaan analysis

23. R  S  R  S  Antibiotic Spectinomycin MIC breakpoint (mg/L)
Disc content
Zone diameter (mm)
R 
S 
R 
S 
Spectinomycin
128 64 25 13 14

24. R  S  R  S  Antibiotic Spectinomycin Ciprofloxacin
MIC breakpoint (mg/L)
Disc content
Zone diameter (mm)
R 
Low R
S 
R 
S 
Spectinomycin
128 - 64 25 13 14
Ciprofloxacin 2
0.12-1
0.06 1
15-28 29
The MIC breakpoint has been lowered to ensure that isolates with reduced susceptibility to ciprofloxacin are detected.

25. R  S  R  S  Antibiotic Spectinomycin Ciprofloxacin Nalidixic acid
MIC breakpoint (mg/L)
Disc content
Zone diameter (mm)
R 
S 
R 
S 
Spectinomycin
128 64 25 13 14
Ciprofloxacin 2
0.12-1
0.06 1
15-28 29
Nalidixic acid - 30
Quinolone resistance is most reliably detected with nalidixic acid. Strains with reduced susceptibility to fluoroquinolones have no zone of inhibition with nalidixic acid.

26. R  S  R  S  Antibiotic Spectinomycin Ciprofloxacin Nalidixic acid
MIC breakpoint (mg/L)
Disc content
Zone diameter (mm)
R 
Low R
S 
R 
S 
Spectinomycin
128 - 64 25 13 14
Ciprofloxacin 2
0.12-1
0.06 1
15-28 29
Nalidixic acid 30
Penicillin
1 unit 17
18-25 26
Test for -lactamase.

27. R  S  R  S  Antibiotic Spectinomycin Ciprofloxacin Nalidixic acid
MIC breakpoint (mg/L)
Disc content
Zone diameter (mm)
R 
Low R
S 
R 
S 
Spectinomycin
128 - 64 25 13 14
Ciprofloxacin 2
0.12-1
0.06 1
15-28 29
Nalidixic acid 30
Penicillin
1 unit 17
18-25 26
Cefuroxime 5 19 20
Ceftriaxone
0.5
0.25 34 35
Resistance to ceftriaxone has not been described. Isolates with chromosomally encoded penicillin resistance (low level) have slightly reduced zones of inhibition with ceftriaxone but remain susceptible. Confirm by MIC.

28. R  S  R  S  Antibiotic Spectinomycin Ciprofloxacin Nalidixic acid
MIC breakpoint (mg/L)
Disc content
Zone diameter (mm)
R 
Low R
S 
R 
S 
Spectinomycin
128 - 64 25 13 14
Ciprofloxacin 2
0.12-1
0.06 1
15-28 29
Nalidixic acid 30
Penicillin
1 unit 17
18-25 26
Cefuroxime 5 19 20
Ceftriaxone
0.5
0.25 34 35
Tetracycline 10
14-26 27
Use tetracycline result to infer susceptibility to doxycycline. Isolates with plasmid-mediated resistance have no zones of inhibition and those with low-level chromosomal resistance have zones mm

29. R  S  R  S  Antibiotic Spectinomycin Ciprofloxacin Nalidixic acid
MIC breakpoint (mg/L)
Disc content
Zone diameter (mm)
R 
Low R
S 
R 
S 
Spectinomycin
128 - 64 25 13 14
Ciprofloxacin 2
0.12-1
0.06 1
15-28 29
Nalidixic acid 30
Penicillin
1 unit 17
18-25 26
Cefuroxime 5 19 20
Ceftriaxone
0.5
0.25 34 35
Tetracycline 10
14-26 27
Erythromycin 11 12
Azithromycin 15 28
Rifampicin 21

30. R  S  R  S  Antibiotic Spectinomycin Ciprofloxacin Nalidixic acid
MIC breakpoint (mg/L)
Disc content
Zone diameter (mm)
R 
Low R
S 
R 
S 
Spectinomycin
128 - 64 25 13 14
Ciprofloxacin 2
0.12-1
0.06 1
15-28 29
Nalidixic acid 30
Penicillin
1 unit 17
18-25 26
Cefuroxime 5 19 20
Ceftriaxone
0.5
0.25 34 35
Tetracycline 10
14-26 27
Erythromycin 11 12
Azithromycin 15 28
Rifampicin 21

31. Next?
Cefixime

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