determination of the antimicrobial susceptibility of neisseria gonorrhoeae trevor winstanley rebecca...
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Determination of the antimicrobial susceptibility of
Neisseria gonorrhoeae
Trevor Winstanley Rebecca Clarke
Department of MicrobiologyRoyal Hallamshire Hospital
Sheffield UK29 July 2003 Freeman Hospital
Gonorrhoea
2nd most common bacterial STD > 22,500 episodes diagnosed in GUM
clinics in England & Wales (2001) Highest incidence
Males 20-24 256/100,000 Females 16-19 198/100,000
Epidemiology Concentrated within demographic
and behavioural risk groups High-levels of
re-infection concurrent STDs asymptomatic infection
Public health concern
Increasing incidence Poor reproductive and sexual
health outcomes High prevalence of resistance
onward transmission adverse clinical sequelae
Antimicrobial resistance 10 -12% of gonococcal isolates are
resistant to some degree inner cities those acquiring infections abroad gay and bisexual men
Plasmid-mediated
ß-lactamase
Tetracycline
PPNG Positive < 16 mg/L
TRNG Negative 16 mg/L
PP/TRNG Positive 16 mg/L
Chromosomal
ß-lactamase
PenicillinTetracyclin
e
PenR Negative 1 mg/L < 2 mg/L
TetR Negative < 1 mg/L 2 - 8 mg/L
CMRNG Negative 1 mg/L 2 - 8 mg/L
Chromosomal
Ciprofloxacin
Intermediate
0.125 < 1 mg/L
Resistant 1 mg/L
Spectinomycin
Resistant 128 mg/L
Ceftriaxone Decreased 0.5 mg/L
Azithromycin Resistant 1 mg/L
U.K. guidelines Easily treatable
95% cure from 1st line therapy Penicillins Fluoroquinolones
ciprofloxacin, ofloxacin Cephalosporins
ceftriaxone, cefixime (Doxycycline / tetracycline)
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
GRASP 2001 (n = 2666)
PenicillinPlasmid 3.0%
Chromosome 5.1%
TetracyclinePlasmid 4.6%
Chromosome 27.9%
CiprofloxacinIntermediate 2.6%
Resistant 3.1%
Azithromycin Resistant 0.3%
SpectinomycinResistant ND
Ceftriaxone
Cure ratesUncomplicated genital gonorrhoeaRecommended dosage
Susceptible > 95% Intermediate 90-95% Resistant < 90%
WHO; SRGA; NCCLS
Media
NCCLS SRGA BSAC ARMRL GRASP
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
Methodology 222 distinct isolates from 5
geographical regions 5 WHO control strains Disc diffusion tests, MICs
BSAC methodology ß-lactamase
Nitrocefin
0
5
10
15
20
25
30
48464442403836343230282624222018161412108642
Zone diameter (mm)
Nu
mb
er
of
iso
late
s
32
16
8
4
2
1
0.5
0.25
0.125
0.064
0.032
0.016
0.008
0.004
MIC(mg/ L)
Penicillin
Amoxycillin
0
5
10
15
20
25
30
464442403836343230282624222018161412108642
Zone diameter (mm)
Nu
mb
er
of
iso
late
s
32
16
8
4
2
1
0.5
0.25
0.125
0.064
0.032
0.016
0.008
0.004
MIC(mg/ L)
Co-amoxyclav
0
5
10
15
20
25
30
50484644424038363432302826242220181614121086
Zone diameter (mm)
Nu
mb
er
of
iso
late
s
32
16
8
4
2
1
0.5
0.25
0.125
0.064
0.032
0.016
0.008
0.004
MIC(mg/ L)
Ciprofloxacin
0
5
10
15
20
25
30
35
585654525048464442403836343230282624222018161412108642
Zone diameter (mm)
Nu
mb
er
of
iso
late
s
32
16
8
4
2
1
0.5
0.25
0.125
0.064
0.032
0.016
0.008
0.004
MIC(mg/ L)
Nalidixic acid
0
5
10
15
20
25
30
35
40
45
50
48464442403836343230282624222018161412108642
Zone diameter (mm)
Nu
mb
er
of
iso
late
s
32
16
8
4
2
1
0.5
0.25
0.125
0.064
0.032
0.016
0.008
0.004
MIC(mg/L)
Tetracycline
0
5
10
15
20
25
30
5048464442403836343230282624222018161412108642
Zone diameter (mm)
Nu
mb
er
of
iso
late
s
16
8
4
2
1
0.5
0.25
MIC (mg/ L)
Metzler & DeHaan analysis
AntibioticMIC breakpoint (mg/L)
Disc content
Zone diameter (mm)
R S R S
Spectinomycin 128 64 25 13 14
Antibiotic
MIC breakpoint (mg/L)Disc
content
Zone diameter (mm)
R Low R S R
Low R S
Spectinomycin 128 - 64 25 13 - 14
Ciprofloxacin 2 0.12-1 0.06 1 14 15-28 29
The MIC breakpoint has been lowered to ensure that isolates with reduced susceptibility to ciprofloxacin are detected.
AntibioticMIC 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 14 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.
Antibiotic
MIC breakpoint (mg/L)Disc
content
Zone diameter (mm)
R Low R S R
Low R
S
Spectinomycin 128 - 64 25 13 - 14
Ciprofloxacin 2 0.12-1 0.06 1 14 15-28 29
Nalidixic acid - - - 30 - - -
Penicillin 2 0.12-1 0.06 1 unit 17 18-25 26
Test for -lactamase.
Antibiotic
MIC breakpoint (mg/L)Disc
content
Zone diameter (mm)
R Low R S R
Low R
S
Spectinomycin 128 - 64 25 13 - 14
Ciprofloxacin 2 0.12-1 0.06 1 14 15-28 29
Nalidixic acid - - - 30 - - -
Penicillin 2 0.12-1 0.06 1 unit 17 18-25 26
Cefuroxime 2 - 1 5 19 - 20
Ceftriaxone 0.5 - 0.25 5 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.
Antibiotic
MIC breakpoint (mg/L)Disc
content
Zone diameter (mm)
R Low R S R
Low R
S
Spectinomycin 128 - 64 25 13 - 14
Ciprofloxacin 2 0.12-1 0.06 1 14 15-28 29
Nalidixic acid - - - 30 - - -
Penicillin 2 0.12-1 0.06 1 unit 17 18-25 26
Cefuroxime 2 - 1 5 19 - 20
Ceftriaxone 0.5 - 0.25 5 34 - 35
Tetracycline 2 - 1 10 13 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 14-26 mm
Antibiotic
MIC breakpoint (mg/L)Disc
content
Zone diameter (mm)
R Low R S R
Low R
S
Spectinomycin 128 - 64 25 13 - 14
Ciprofloxacin 2 0.12-1 0.06 1 14 15-28 29
Nalidixic acid - - - 30 - - -
Penicillin 2 0.12-1 0.06 1 unit 17 18-25 26
Cefuroxime 2 - 1 5 19 - 20
Ceftriaxone 0.5 - 0.25 5 34 - 35
Tetracycline 2 - 1 10 13 14-26 27
Erythromycin 1 - 0.5 5 11 - 12
Azithromycin 2 - 1 15 27 - 28
Rifampicin 2 - 1 2 20 - 21
Antibiotic
MIC breakpoint (mg/L)Disc
content
Zone diameter (mm)
R Low R S R
Low R
S
Spectinomycin 128 - 64 25 13 - 14
Ciprofloxacin 2 0.12-1 0.06 1 14 15-28 29
Nalidixic acid - - - 30 - - -
Penicillin 2 0.12-1 0.06 1 unit 17 18-25 26
Cefuroxime 2 - 1 5 19 - 20
Ceftriaxone 0.5 - 0.25 5 34 - 35
Tetracycline 2 - 1 10 13 14-26 27
Erythromycin 1 - 0.5 5 11 - 12
Azithromycin 2 - 1 15 27 - 28
Rifampicin 2 - 1 2 20 - 21
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