1. Copyright © 2016 Cian Foley cfoley@nmh.ie
Neisseria gonorrhoeae is an obligate human pathogen which causes infections such as urethritis, cervicitis, pelvic
inflammatory disease (PID), anorectal infections, pharyngitis, conjunctivitis, bloodstream infection, meningitis, and septic
arthritis[1,2].
Severe complications associated with gonococcal infections can increase the rate of transmission of other pathogens,
such as HIV and other STIs, due to shared transmission routes; impairment of the immune response; tissue damage due
to elevated cytokine levels and subsequent inflammation; and breaches in the mucosal epithelium[2,3,4].
Antimicrobial resistance (AMR) among Neisseria gonorrhoeae is a worldwide public health threat.Test of Cure (TOC) is
recommended by Public Health England (PHE) in the UK for monitoring of AMR in N. gonorrhoeae[5]. N. gonorrhoeae must
be cultured to detect AMR but they are labile organisms which survive poorly in the environment. Furthermore, many
hospitals and clinics transport swabs to off-site laboratories, prolonging the pre-analytical time.
To determine optimal pre-analytical and analytical conditions to maximise culture of N. gonorrhoeae –pre-analytical time
before inoculation and incubation, swab type used, temperature for storage and agar used for culture.
Twenty nine stored clinical isolates of Neisseria gonorrhoeae were tested. A 0.5 McFarland concentration of each isolate
was made using distilled H2O (dH2O) and doubling dilutions were performed to a factor of 16 (1:65636). 1 l from each
dilution was inoculated onto Columbia Chocolate agar (CCA) [LIP Diagnostics, Fannin Ltd. Galway, Ireland] and
incubated at 35oC +/- 2oC in a 5% CO2 atmosphere for 48 hours. Both dry cotton swabs and Transwab® Amies Charcoal
swabs [MWE. Corsham, UK] were inoculated using a high concentration (0.5 McFarland) and a low concentration
(determined via dilutions) for each isolate plus a control, N. gonorrheoae ATCC 49226, and stored for varying lengths of
time (2, 6, 12, 24, 36, 48, & 72 hours) and at 2 temperatures (room temperature (~20-22oC) and 4oC). Each swab was
then inoculated onto both Columbia Blood agar (CBA) [LIP Diagnostics, Fannin Ltd. Galway, Ireland] and CCA and
incubated for 48 hours at 35oC +/- 2oC in an aerobic atmosphere and a 5% CO2 atmosphere, respectively.All tests were
performed in duplicate.
• Fifteen isolates were recovered after 12 hours delay on CBA compared to twenty eigth on CCA
when using optimal conditions.
• After longer storage at 36, 48, and 72 hours, recovery of N . gonorrhoeae at 4oC was superior to RT
(p<0.0001). Seventeen (59%) high concentration isolates grew after 48 hours and fifteen (52%)
after 72 hours at 4oC.
• From low concentrations, there was a significant reduction in the recovery of N . gonorrhoeae after
2 hours storage and few isolates recovered after 12 hours at both RT and 4oC.
• High concentrations of GC i.e. new active infections, can be cultured easily up to a 6 hour delay
before inoculation, if either a dry swab or a charcoal swab is used and if storage is at RT or 4oC, so
long as the swab is inoculated onto chocolate agar and not blood agar.
• If there is >6 hours before inoculation of the swab, it is essential that charcoal swabs are used, as
dry swabs no longer support the growth of GC.
• If there is >12 hours before inoculation of the swab, it is essential to store the swab at 4oC.
• Low concentrations of GC (e.g. post-treatment TOC) grow poorly under all conditions tested,
even with a 2 hour delay in inoculation. For TOC swabs, either direct inoculation at the clinic with
rapid incubation or PCR should be used.
References: 1. Mayor et al. (2012) Diagnosis and Management of Gonococcal Infections. American Academy of Family Physicians. 86(10): 931-8. 2. Lewis (2010) Will Targeting Oropharyngeal Gonorrhoea Delay the Further Emergence of Drug-Resistant Neisseria
gonorrhoeae Strains? Sex Transm Infect. 0: 1-4. 3. Cheng et al. (2014) Changes in the Six Mosy Common Sequence Types of Neisseria gonorrhoeae, including ST4378, Identified by Surveillance of Antimicrobial Resistance in Northern Taiwan from 2006 – 2013. J
Microbiol. 4. Marschalkó et al. (2015) Sexually Transmitted Coinfections. HIV Coinfections. Orv Hetil. 5. Public Health England (2015) Guidance for the Detection of Gonorrhoea in England.Available at:
http://www.gov.uk/government/uploads/system/uploads/attachment_data/file/405293/170215_Gonorrhoea_testing_guidance_REVISED_2_.pdf
Optimal Pre-Analytical and Analytical Conditions for Culture of Neisseria gonorrhoeae
Cian Foley, Susan Knowles, Anya Curry, National Maternity Hospital, Dublin 2, Ireland
Introduction
Objectives
Materials and Methods
Results
Conclusion
Table 1. Recovery rates (%) of isolates A) room temperature v 4oC B) Transwab®
Amies Charcoal swabs v dry cotton swabs C) Chocolate agar v Blood agar
• All isolates survived better on Transwab® Amies Charcoal swabs compared with dry cotton swabs at all time points
(p<0.001) e.g. 28/29 isolates were recovered after 24 hours delay compared to 1/29 recoverable from dry cotton
swabs when using CCA at 4oC.
• Recovery of N. gonorrhoeae was also superior when cultured onto CCA, although CBA supported the growth of the
majority of N. gonorrhoeae isolates.
B)A)
C)