6. Neisseria Species

6. Application of the CDS to Neisseria Sp.

6.1       Preliminary Testing by the Routine Laboratory

Please note: This section is intended to encompass the preliminary testing of Neisseria species by routine diagnostic laboratories. Those laboratories that need to carry out more detailed examinations on either Neisseria meningitidis or Neisseria gonorrhoeae should consult section 6.2 on Neisseria provided by Prof Lahra. The majority of countries where the CDS is used have highly specialised public health laboratories that carry out detailed antibiotic susceptibility and other studies on both Neisseria meningitidis and Neisseria gonorrhoeae. It is important that isolates of both these species are sent without delay to the appropriate reference centre.

  • Neisseria meningitidis

If the routine laboratory has ready access to a reference laboratory who can provide a rapid turnaround time they may elect to forgo testing. Otherwise susceptibility testing is performed on blood Sensitest Agar (Section 2.2.1.) and incubated at 35‑36°C, in 5% CO2. Invasive isolates of N. meningitidis should be sent to a reference centre for serotyping and confirmation of identity and antibiotic susceptibilities. The antibiotics calibrated for Neisseria meningitis are shown in Table 12.1.b.

  • Neisseria meningitidis and benzylpenicillin

Benzylpenicillin 0.5 u (P 0.5 u) has been calibrated for the testing of N. meningitidis and the annular radius of the zone of inhibition for susceptible strains is ≥ 4 mm. The MIC of benzylpenicillin for susceptible strains is ≤ 0.25 mg/L.

Note 1: Some authorities recommend a susceptible breakpoint of ≤ 1 mg/L, however the “susceptible” breakpoint of < 0.25 mg/L for penicillin was established with wild type isolates of Neisseria meningitidis and correlated well with clinical response. Strains with a diminished susceptibility to penicillin appeared after the initial calibrations and there is a dearth of strong clinical evidence to indicate that infections with these strains would respond to treatment with penicillin. This should be kept in mind when interpreting susceptibility test reports of other methods where the susceptible breakpoint is higher than 0.25 mg/L.

Note 2: More recently the Neisseria Reference laboratory has raised an issue regarding the accuracy of the CDS Test in Testing N. meningitis to penicillin. This is contrary to the strong evidence of the CDS Laboratory which demonstrates a highly satisfactory separation of N. meningitis into Resistant and Susceptible. Until this is resolved laboratories may chose to either perform and report the results of the test or advise the clinician to continue with empirical treatment.

  • Neisseria gonorrhoeae

Disc susceptibility testing by the routine laboratory is inappropriate for two reasons. First susceptibility testing of Neisseria gonorrhoeae is a complex procedure more appropriately performed by a specialised laboratory.  Secondly, clinical practice is not to await results of antibiotic susceptibility testing before initiating treatment. The choice of antibiotic is made empirically and based on the epidemiological data gathered by the reference centre.

 

Note 1 Section 6.2 below is supplied in its entirety by Prof. Monica Lahra of the Neisseria Reference Laboratory, Microbiology Department, Randwick. Comments or questions about the contents of this section should be addressed to the Neisseria Reference Laboratory.

Note 2 In section 6.2 the results of “CDS testing” are reported in at least 3 categories based on zone sizes. Strictly speaking these tests are not CDS Tests where, by definition, it only reports zones sizes dichotomously as susceptible or resistant. For the sake of expediency some licence has been granted to the reference laboratory to modify the CDS in this way but this does not indicate support for the wider application of this modification or any other modification outside the reference laboratory.

6.2  Testing by the Reference Laboratory

Application of the CDS to Neisseria gonorrhoeae and Neisseria meningitidis

This section is intended to provide a guide for testing and interpreting antimicrobial susceptibility testing in a diagnostic setting for antibiotics clinically relevant to the treatment of gonococcal and meningococcal disease.

Laboratory surveillance to monitor antimicrobial resistance in N. gonorrhoeae and N. meningitidis from invasive meningococcal disease cases is performed by National Neisseria Network (NNN): the Australian Gonococcal Surveillance Programme (AGSP)1, and the Australian Meningococcal Surveillance Programme (AMSP)2, The NNN is a collaboration of jurisdictional reference laboratories, in co-operation with private and public sector laboratories3. For epidemiological and public health reasons, laboratories are requested to refer all N. gonorrhoeae isolates to the appropriate jurisdictional Neisseria Reference Laboratory.

METHODS

Minimum Inhibitory Concentration

The determination of Minimum Inhibitory Concentration (MIC) by the agar plate dilution method is the gold standard for determining the category of susceptibility of N. gonorrhoeae and N. meningitidis3,4.

The CDS-applied Etest® MIC method5 has been validated by the WHO Collaborating Centre for STD, and Neisseria Reference Laboratory, Sydney. For a link to this method see http://www.sciencedirect.com/science/article/pii/S0732889316300955.

CDS Disc Diffusion Method

CDS testing of N. gonorrhoeae and N. meningitidis using the AGSP and AMSP method and interpretive criteria is performed as described in section 2.2, with the modification of Chocolate Columbia Blood Agar as the testing media and incubated 18-24 hours at 36°C ± 1°C in 5% CO2 in air with > 80% humidity. The annular radius (AR) of the zone of inhibition of growth around the antibiotic disc is then measured and reported according to the AGSP and AMSP interpretive criteria.

INTERPRETATIVE CRITERIA FOR N. GONORRHOEAE

Table 1: AGSP Interpretative Criteria3,6,7 for N. gonorrhoeae MIC values.

Antibiotic MIC (mg/L) Sensitive Less sensitive a Resistant
Ceftriaxone <0.06 0.06-0.25 Not defined b
Azithromycin <1.0 Not defined ≥ 1.0 c
Penicillin <0.06 0.06 – 0.5 >0.5
Ciprofloxacin <0.06 0.06-0.5 >0.5
Gentamicin <8 8-16 >16
Spectinomycin ≤64 Not defined >64

Table 1 Notes:

a The term used to describe intermediate susceptibility in ceftriaxone is “decreased susceptibility”.

b The absence or rare occurrence of an adequate number of evidence-based correlates between the MIC of isolates and treatment outcome means the breakpoint for resistance cannot yet be determined. 8, 9

c The Centre for Disease Control (CDC), Atlanta, USA states that in the absence of established criteria, the use of critical MICs ≥1.0mg/L to interpret the susceptibility of N. gonorrhoeae to this agent is recommended until more extensive assessments of clinical treatment outcome to this agent is available. 4

 The annular radius parameters used for testing N. gonorrhoeae susceptibility and their indicative MIC and category of susceptibility are shown in tables 2 and 3:

Table 2: Antibiotic annular radius and indicative susceptibility and for N.gonorrhoeae

 

Antibiotic & Disc Potency Annular Radius Indicative MIC (mg/L) Indicative Category

Ceftriaxone

0.5μg

≥10mm <0.06 Susceptible
≤9mmd 0.06 – 0.25 Decreased Susceptibility d

Azithromycin

15μg

 

>6mme <1.0 Susceptible
≤6mme ≥1.0 Resistant f

Penicillin

0.5U

> 9mm <0.06 Susceptible
3 – 9mmg 0.06 – 0.50 g Less Susceptible
<3mmg >0.5 g Resistant

Gentamicin

30 μg

≥ 6 mm ≤ 4 Susceptible
<6 mm h >4 h See comment h

Spectinomycin

100μg

≥ 6 mm <128 Susceptible
<6 mm ≥128 Resistant i

Table 3: Ciprofloxacin annular radius and indicative susceptibility and for N.gonorrhoeae

Antibiotic

Annular radius

Ciprofloxacin

1 µg

Annular radius

Nalidixic acid

30 µg

Indicative

MIC (mg/L)

Susceptibility Category

 

Ciprofloxacin j

> 11mm > 6mm <0.06 Susceptible
6 –11mm ≤ 6 mm 0.06 – 0.5

Less

Susceptible

  ≤ 6mm ≤ 6mm >0.5 Resistant

Tables 2 and 3 Notes:

d Internal validations have shown that using the ceftriaxone 0.5μg disc, N. gonorrhoeae isolates with AR of 5-9mm have an MIC value in the range of 0.016-0.125mg/L. Determination of ceftriaxone MIC is required to definitively assign the category of susceptibility. Isolates with suspected decreased susceptibility or resistance to ceftriaxone should be referred to the appropriate jurisdictional Neisseria Reference Laboratory for MIC testing.

e Internal validations have shown that a small proportion of isolates (5-10%) that have an AR of the inhibitory zone to azithromycin 15 μg disc of 6.0-7.0mm have an azithromycin MIC ≥ 1.0mg/L. It is therefore recommended that strains with AR of the inhibitory zone to azithromycin 15 μg disc of ≤7.0mm should have the azithromycin MIC value determined and the susceptibility category confirmed.

 f The Centre for Disease Control (CDC), Atlanta, USA state that in the absence established criteria, the use of critical MICs ≥1.0mg/L to interpret the susceptibility of N.gonorrhoeae to this agent is recommended until more extensive assessments of clinical treatment outcome to this agent is available. 4

 g Isolates with AR close to the cut off for less sensitive/resistant category (i.e. 2-4mm) may have an MIC that is 1 standard two-fold dilution below the MIC resistant breakpoint. The susceptibility category of such isolates should be confirmed by MIC testing and referred to the appropriate jurisdictional Neisseria Reference Laboratory.

h Internal validations have shown that using the gentamicin 30μg disc, N. gonorrhoeae isolates with AR of 2-5mm have an MIC value in the range of 4-16 mg/L. The absence or rare occurrence of an adequate number of evidence-based correlates between the AR of isolates with an MIC value of ≥ 8 mg/L and treatment outcome means the AR breakpoint and indicative category of susceptibility cannot yet be determined with certainty. Determination of gentamicin MIC is required to definitively assign the category of susceptibility. Isolates with suspected intermediate susceptibility or resistance to gentamicin should be confirmed by MIC testing and referred to the appropriate jurisdictional Neisseria Reference Laboratory.

i Resistance observed to spectinomycin is rare. Any isolates suspected to have spectinomycin resistance should be confirmed by MIC testing and referred to the appropriate jurisdictional Neisseria Reference Laboratory.

j Testing is performed using a combination of both ciprofloxacin 1 µg and nalidixic acid 30 µg discs. The category of susceptibility for ciprofloxacin is derived by considering the annular radius measurements obtained with both antibiotic discs.

 

INTERPRETATIVE CRITERIA FOR N. MENINGITIDIS

Table 4: AMSP interpretative criteria2,3 of MIC for N.meningitidis

Antibiotic MIC (mg/L) Sensitive Less Sensitive Resistant
Ceftriaxone ≤0.25 Not definedk Not definedk
Penicillin <0.06 0.06 – 0.5 >0.5
Ciprofloxacin <0.06 0.06-0.5 >0.5
Rifampicin ≤0.5 Not defined >0.5

Table 4 Notes:

k The absence or rare occurrence of isolates with MIC values above the susceptible breakpoint are very rare or not yet reported. The absence or rare occurrence of resistant strains precludes defining any result categories other than sensitive10.

The annular radius parameters for the various antibiotics used for testing N. meningitidis susceptibility and their indicative MIC and category of susceptibility are shown in tables 5 and 6:

Table 5: Antibiotic annular radius and indicative susceptibility and for N.meningitidis

 

Antibiotic Annular Radius

Indicative

MIC (mg/L)

Susceptibility Category

Ceftriaxone

0.5μg

≥9 mm ≤0.25 Susceptible
≤8 mm Refer to reference lab for MIC testing l
Penicillin See notesm See notesm See notesm

Rifampicin

1μg

≥ 6 mm <1 Susceptible
<6 mm ≥1 Resistantn

 

Table 6: Ciprofloxacin annular radius and indicative susceptibility and for N.meningitidis

 

Antibiotic

Annular radius

Ciprofloxacin

1 µg

Annular radius

Nalidixic acid

30 µg

Indicative

MIC (mg/L)

Susceptibility Category

 

Ciprofloxacin o

> 11 mm > 6 mm <0.06 Susceptible
6 –11 mm ≤ 6 mm 0.06 – 0.5 Less susceptible
≤ 6 mm ≤ 6 mm >0.5 Resistant

Tables 5 and 6 Notes:

l The absence or rare occurrence of an adequate number of evidence-based correlates between the MIC of isolates and treatment outcome means the indicative MIC cannot be stated with any degree of certainty. The susceptibility category of such isolates should be confirmed by MIC testing and referred to the appropriate jurisdictional Neisseria Reference Laboratory10.

m Penicillin disc diffusion testing, using the CDS method and chocolate agar, is not recommended to determine the antimicrobial susceptibility category of N. meningitidis to penicillin. Internal validation testing of N. meningitidis against penicillin 0.5U disc produced results with a high percentage interpretive susceptibility category error, particularly in strains that had an MIC equivalent at close to the various category breakpoints. This has been reported elsewhere2,17. MIC determination by Etest® or agar dilution is required for antimicrobial susceptibility categorisation.

n Isolates with a rifampicin annular radius measurement of <6mm should be referred for MIC testing. Isolates with a rifampicin annular radius measurement of >2mm but <6 mm should be referred for MIC testing. Resistance to rifampicin is uncommon. The number of isolates available for testing with a rifampicin MIC ≥ 0.5 in internal validation studies is small. The significance of rifampicin MIC ≥ 1mg/L but <100mg/L is not certain, however, the breakpoint for resistance is considered by the AMSP to be ≥1mg/L2,3,12.

 o Testing is performed using a combination of both ciprofloxacin 1 µg and nalidixic acid 30 µg discs. The category of susceptibility for ciprofloxacin is derived by considering the annular radius measurements obtained with both antibiotic discs.

 

INTENDED USE OF REFERENCE STRAINS AND QUALITY CONTROL

The World Health Organisation (WHO) reference strains are those recommended for antimicrobial susceptibility testing of N. gonorrhoeae8,13 and N. meningitidis. To reduce the risk of exposure, the use of N. meningitidis control strains is not recommended.

The WHO reference strains are available from the WHO Collaborating Centre for STD, and Neisseria Reference Laboratory, Sydney.

Tel: +61 293829084; Email: NSWPATH-WHOCCSYDNEY@health.nsw.gov.au

Table 7: WHO N. gonorrhoeae reference strains recommended for QC and acceptable annular radius measurement ranges.

 

Antibiotic &

Disc Potency

Reference Strain

Mean A.R.

(Range)

Expected MIC range (mg/L)

Susceptibility

Category

 

Ceftriaxone

0.5μg

 

WHO C

9.5mm

(8.3 – 10.6)

0.008-0.032 Susceptible
WHO L

5.5mm

(4.4 – 6.7)

0.064-0.25 Decreased Susceptible

 

Azithromycin

15μg

 

WHO C

10mm

(8.3 – 11.3)

0.064-0.25 Susceptible
WHO P

5.5mm

(4.2 – 7.0)

1.0-4.0 Resistant

 

Penicillin

0.5U

WHO C

3mm

(2.1 – 4.7)

0.25-1.0

Less

Susceptible

WHO L 0mm 1.0-4.0 Resistant

 

Spectinomycin 100μg

 

WHO C

8mm

(6.7-9.1)

<64 Susceptible
WHO O 0mm >1024 Resistant

 

Ciprofloxacin

1μg

WHO C

14mm

(12.5 – 16.0)

0.004-0.016 Susceptible
WHO L 0mm ≥32 Resistant

 

Nalidixic Acid

30μg

WHO C

13mm

(11.0 – 14.0)

Susceptible
WHO L 0mm Resistant


References

 

1       J. W. Tapsall et al. Australian gonococcal surveillance programme. Penicillin sensitivity of gonococci in Australia: development of an Australian gonococcal surveillance programme. Br J Vener Dis 1984;60:226-230

2       Tapsall, J.W. 1997. Annual report of the Australian Meningococcal Surveillance Programme, 1997. Communicable Diseases Intelligence, Volume 22, Issue number 10 – 1 October 1998

3       Tapsall, J. and members of the National Neisseria Network of Australia. 2004. Antimicrobial testing and applications in the pathogenic Neisseria. In: Antimicrobial Susceptibility testing methods and practice with an Australian perspective. Ed: Merlino J .Australian Society for Microbiology, Antimicrobial Special Interest Group. Chapter 8, 175-188

4       Centers for Disease Control and Prevention Bulletin B88. 2005. Neisseria Gonorrhoeae Reference Strains for Antimicrobial Susceptibility Testing. Web site: http://www.cdc.gov/std/Gonorrhea/arg/B88-Feb-2005.pdf

5       Enriquez, R.P., Goire, N., Kundu, R., Gatus, B., Lahra, M M. 2016. A comparison of agar dilution with the Calibrated Dichotomous Sensitivity (CDS) and Etest methods for determining the minimum inhibitory concentration of ceftriaxone against Neisseria gonorrhoeae. J Diagn Microbiol Infect Dis. Volume 86, Issue 1, September 2016, 40-43

6       Lahra, M M, Enriquez, R. 2015. Australian Gonococcal Surveillance Programme, 1 January to 31 March 2015. Communicable Diseases Intelligence Volume 39 No 2 – June 2015

7       Bala, M., Singh, V., Philipova, I., Bhargava, A., Joshi, C., Unemo, M. 2016. Gentamicin in vitro activity and tentative gentamicin interpretation criteria for the CLSI and calibrated dichotomous sensitivity disc diffusion methods for Neisseria gonorrhoeae. J. Antimcrob Chemoth. 2016 Jul;71(7):1856-9

8       Unemo, M et al. Laboratory Diagnosis of Sexually Transmitted Infections, including human immunodeficiency virus. World Health Organisation bulletin 2013 pp38-53 Web site: http://www.who.int/reproductivehealth/publications/rtis/9789241505840/en/

9       World Health Organisation bulletin 2012 Global Action Plan to Control the Spread and Impact of Antimicrobial Resistance in Neisseria gonorrhoeae. Web site: http://www.who.int/reproductivehealth/publications/rtis/9789241503501/en/

10     Centers of Disease Control and Prevention Laboratory Methods for the Diagnosis of Meningitis 2nd Ed. 2011. Web site:

http://www.cdc.gov/meningitis/lab-manual/chpt11-antimicrobial-suscept-testing.html

11     Jorgensen, J.H. et al. Multilaboratory Evaluation of Disk Diffusion Antimicrobial Susceptibility Testing of Neisseria meningitidis Isolates. Journal of Clinical Microbiology, Vol44, No.5 2006.

12     Tapsall, J.W. 2008. Annual report of the Australian Meningococcal Surveillance Programme, 2008. Communicable Diseases Intelligence Volume 33 No 3 – September 2009

13     Unemo, M., Fasth, O., Fredlund, H., Limnios, A., Tapsall, J. 2009. Phenotypic and genetic characterization of the 2008 WHO Neisseria gonorrhoeae reference strain panel intended for global quality assurance and quality control of gonococcal antimicrobial resistance surveillance for public health purposes. J. Antimcrob Chemoth. 63, 1142-1151