6.Application of the CDS to Neisseria sp.
6.1. Preliminary Testing by the Routine Laboratory
Please note: Section 6.1 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 Monica 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, Neisseria meningitidis 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 11.1.b. The morbidity and mortality of meningitis increases dramatically with delay in initiation of antibiotic treatment therefore empiric therapy (usually with a combination of agents) based on sound clinical experience is the accepted practice in the management of patients with meningitis. Antibiotic susceptibility testing of the infecting organism plays very much a secondary role in the management of meningitis.
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. The CDS disc test was recently reappraised and found to be highly specific in reporting only strains with an MIC of ≤ 0.25 mg/L as susceptible. This breakpoint continues to be supported by a number of clinical recommendations for treatment1,2.
Neisseria gonorrhoeae
Disc susceptibility testing by the routine laboratory is not recommended 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: Section 6.2 below is supplied in its entirety by Prof. Monica Lahra of the World Health Organisation Collaborating Centre for STD and Neisseria Reference Laboratory, Microbiology Department, Randwick. Comments or questions about the contents of this section should be addressed to NSWPATH-WHOCCSYDNEY@health.nsw.gov.au.
6.2. Testing by the Reference Laboratory
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 disease.
Laboratory surveillance to monitor antimicrobial resistance in N. gonorrhoeae is performed by Neisseria References Laboratories in each jurisdiction in Australia (National Neisseria Network (NNN) for the Australian Gonococcal Surveillance Programme (AGSP)3 and Australian Meningococcal Surveillance Programme (AMSP)4. The NNN is a collaboration of jurisdictional reference laboratories, in co-operation with private and public sector laboratories5. For epidemiological and public health reasons, laboratories are requested to refer all N. gonorrhoeae and invasive N. meningitidis isolates to the appropriate jurisdictional Neisseria Reference Laboratory.
6.2.1. Methods
Application of the CDS to Neisseria gonorrhoeae
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. meningitidis5,6.
For N. gonorrhoeae the CDS-applied Etest® MIC method7 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 using the AGSP 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 interpretive criteria.
6.2.2. Interpretative Criteria for N. gonorrhoeae
Table 1: AGSP Interpretative Criteria8,9 for N. gonorrhoeae MIC values.
| Antibiotic MIC (mg/L) | Sensitive | Less sensitivea | Resistant |
| Ceftriaxone | <0.06 | 0.06-0.25 | Not definedb |
| Azithromycin | <1.0 | Not defined | ≥ 1.0c |
| 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 |
| Tetracycline | <0.5 | 0.5 – 1.0 | >1.0 |
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 determined10,11.
c The Centre for Disease Control (CDC), Atlanta, USA states that in the absence of established criteria, the use of critical MICs ≥ 1.0 mg/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 available6.
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 | Decreased Susceptibilityd | |
|
Azithromycin 15μg |
>6mme | <1.0 | Susceptible |
| ≤6mme | ≥1.0 | Resistantf | |
|
Penicillin 0.5U |
> 9mm | <0.06 | Susceptible |
| 3 – 9mmg | 0.06 – 0.50g | Less Susceptible | |
| <3mmg | >0.5g | Resistant | |
|
Gentamicin 30 μg |
≥6mm | ≤ 4 | Susceptible |
| <6 mmh | >4h | See commenth | |
|
Spectinomycin 100μg |
≥6 mm | <128 | Susceptible |
| <6 mm | ≥128 | Resistanti | |
|
Tetracycline 10 μg |
<4 mm | ≥2 | Resistant |
| 4-8 mm | See commentj | See commentj | |
| >8 mm | ≤0.25 | Susceptible |
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 |
| Ciprofloxacink | > 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.6
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 above or 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 30 μg 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 Internal validations have shown that using the tetracycline 10 μg disc, N. gonorrhoeae isolates with AR of 4-8 mm have an MIC value in the range of 0.25-2.0mg/L. Determination of tetracycline MIC is required to definitively assign the category of susceptibility of isolates with an AR in this range. Isolates with suspected intermediate susceptibility or resistance to tetracycline should be confirmed by MIC testing and referred to the appropriate jurisdictional Neisseria Reference Laboratory.
k 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.
6.2.2. Intended use of reference strains and quality control
The World Health Organisation (WHO) reference strains are those recommended for antimicrobial susceptibility testing of N. gonorrhoeae10,12.
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 4: 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 |
ATCC 49226 |
9mm (8.1 – 10.0) |
0.004-0.016 | Susceptible |
| WHO L |
5.5mm (4.4 – 6.7) |
0.125-0.50 | Decreased Susceptible | |
|
Azithromycin 15μg |
ATCC 49226 |
9.5mm (7.7 – 11.5) |
0.25-1.0 | Susceptible |
| WHO P |
5.5mm (4.2 – 7.0) |
2.0-8.0 | Resistant | |
|
Penicillin 0.5U |
ATCC 49226 |
3mm (2.4 – 3.5) |
0.25-1.0 |
Less Susceptible |
| WHO L | 0mm | 1.0-4.0 | Resistant | |
|
Spectinomycin 100μg |
ATCC 49226 |
8.5mm (6.6-10.1) |
8-32 | Susceptible |
| WHO O | 0mm | >1024 | Resistant | |
|
Tetracycline 10 μg |
ATCC 49226 |
6 mm (5.4 – 7.2) |
0.25-1.0
|
Less Susceptible |
| WHO G | 0mm | ≥32 | Resistant | |
|
Ciprofloxacin 1μg |
ATCC 49226 |
15mm (13.3 – 16.4) |
0.001-0.008 | Susceptible |
| WHO L | 0mm | ≥32 | Resistant | |
|
Nalidixic Acid 30μg |
ATCC 49226 |
12.5mm (11.3 – 13.3) |
– | Susceptible |
| WHO L | 0mm | – | Resistant |
References
- NSW Health Guideline. Infants and Children: Acute Management of Bacterial Meningitis: Clinical Practice Guideline 15 Jul 2014. Available at: http://www1.health.nsw.gov.au/pds/ActivePDSDocuments/GL2014_013.pdf
2 Severe sepsis and septic shock: Neisseria meningitidis (Meningococcal sepsis) [revised 2014]. In: eTG complete [Internet}. Melbourne: Therapeutic Guidelines Limited; 2015 Jul. Available at: https://tgldcdp.tg.org.au.acs.hcn.com.au/viewTopic?topicfile=severe-sepsis&guidelineName=Antibiotic#toc_d1e1486 .
- Tapsall, J. W., Cossins, Y.M., Murphy, D.M., Mallon, R., Macleod, C., Raby, J., et al.1984 Australian gonococcal surveillance programme. Penicillin sensitivity of gonococci in Australia: Development of an Australian gonococcal surveillance programme. Br J Vener Dis. 60, 226-230.
4 Tapsall, J.W. 1997. Annual report of the Australian Meningococcal Surveillance Programme, 1997. Communicable Diseases Intelligence, Volume 22, Issue number 10 – 1 October 1998.
5 Tapsall, J. and Members of the National Neisseria Network of Australia. 2004. Antimicrobial testing and applications in Pathogenic Neisseria. Antimicrobial Susceptibility testing methods and practices with an Australian perspective. Australian Society for Microbiology, Antimicrobial Special Interest Group. Chapter 8, 175-186
6 Centers for Disease Control and Prevention Bulletin B88. 2005. Neisseria Gonorrhoeae Reference Strains for Antimicrobial Susceptibility Testing. Available at: http://www.cdc.gov/std/gonorrhea/arg/B88-Feb-2005.pdf
7 Enriquez, R.P., Goire, N., Kundu, R., Gatus, B.J. and 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. Diagn Microbiol Infect Dis. 86, 40-43
8 Lahra, M. M. and Enriquez, R.P.2017. Australian Gonococcal Surveillance Programme, 1 July to 30 September 2016. Communicable Diseases Intelligence Volume 41 No 1, 109-110.
9 Bala, M., Singh, V., Philipova, I., Bhargava, A., Joshi, C. and 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 Chemother. 71, 1856-59
10 Unemo, M., Ballard. R., Ison, C., Lewis, D., Ndowa, F. and Peeling, R. 2013. Laboratory Diagnosis of Sexually Transmitted Infections, including human immunodeficiency virus. World Health Organisation Manual. 38-53 Available at: http://www.who.int/reproductivehealth/publications/rtis/9789241505840/en/
11 World Health Organization (WHO), Department of Reproductive Health and Research. 2012. Global action plan to control the spread and impact of antimicrobial resistance in Neisseria gonorrhoeae, p 1–36. WHO, Geneva, Switzerland. Available at: http://www.who.int/reproductivehealth/publications/rtis/9789241503501/en/
12 Unemo, M.., Golparian. D., Sanchez-Buso. L., Grad. Y., Jacobsson.S. and Ohnish, M. 2016. The novel 2016 WHO Neisseria gonorrhoeae reference strains for global quality assurance of laboratory investigations: phenotypic, genetic and reference genome characterization. J Antimicrob Chemother. 71, 3096-108