David Whiley QPID Laboratory, QCMRI and SASVRC, Children’s Health Service District, and The University of Queensland, Brisbane, Australia.

XDR Neisseria gonorrhoeae: where are we at?

Background –

• Neisseria gonorrhoeae is the etiological agent of the STD gonorrhoea. • Infects mucus membranes: endocervix, urethra, rectum, pharynx and conjunctiva. • Disease:

Females: major cause of pelvic inflammatory disease and may lead to tubal infertility, ectopic pregnancy, and chronic pelvic pain.

Males: primarily causes urethritis. • Globally, disease burden is highest in low income settings. • WHO estimates: approx. 100 million cases of gonorrhoea globally. • Rates are increasing (13,500 notifications in Aust. in 2012)

Gonorrhoea notification rates Australia (1991 to 2012) (per 100,000 population)

1991 1998 2005 2012 0

20

40

60

Background –

Neisseria gonorrhoeae has developed resistance to multiple classes of antimicrobials:

1930 1940 1950 1960 1970 1980 1990 2000 2010

Sulphonamides

Penicillins

Tetracyclines

Spectinomycin Azithromycin

Ciprofloxacin Cefixime

Ceftriaxone

Sulphonamides Ciprofloxacin Cefixime

Ceftriaxone

Penicillins (chromosomally-

mediated)

Penicillins (plasmid-mediated)

Tetracyclines (plasmid-mediated)

Azithromycin Spectinomycin

Antibiotic first reported for treatment of gonorrhoea

Evidence of resistance

Background –

Neisseria gonorrhoeae has developed resistance to multiple classes of antimicrobials:

Extended-spectrum cephalosporins –

• Cefixime no longer considered suitable for use in many regions. (Maldonado & Takhar, Ann Emerg Med. 2013 Jan;61(1):91-5.)

• Increasing numbers of gonococci with reduced-susceptibility to ceftriaxone observed worldwide (including Australia) over several years.

• Late 2010. the first ceftriaxone resistant-N. gonorrhoeae clinical isolate, now named H041, was observed in Japan.

(Ohnishi et al. Emerg Infect Dis. 2011 Jan;17(1):148-9.)

• 2011. Ceftriaxone-resistant N. gonorrhoeae in France; now named F89. (Unemo et al. AAC. 2012 Mar;56(3):1273-80.)

?!

Australian wild-type strains previously: Isolates now generally: Australian strains with reduced susceptibility:

< 0.001 mg/L 0.008 mg/L 0.016 mg/L 0.03 mg/L 0.06 mg/L 0.125 mg/L

Ceftriaxone-resistant NG – Increasing ceftriaxone MICs over time:

2006 2008 2007 2009 2010 2011 0%

2%

4%

6%

2012

NG with reduced-susceptibility (2006-2012) – Australia.

Australian wild-type strains previously: Isolates now generally: Australian strains with reduced susceptibility:

< 0.001 mg/L 0.008 mg/L 0.016 mg/L 0.03 mg/L 0.06 mg/L 0.125 mg/L

Ceftriaxone-resistant NG – Increasing ceftriaxone MICs over time:

Vict ACT NSW Qld WA SA 0%

2%

4%

6%

NT Tas

NG with reduced-susceptibility (2011) – Australia.

Australian wild-type strains previously: Isolates now generally: Australian strains with reduced susceptibility:

< 0.001 mg/L 0.008 mg/L 0.016 mg/L 0.03 mg/L 0.06 mg/L 0.125 mg/L

Ceftriaxone-resistant NG – Increasing ceftriaxone MICs over time:

NB: Technically still sensitive & no reported treatment failures for genital gonorrhoea in Australia.

HOWEVER: Increasing numbers of ceftriaxone treatment

failure for pharyngeal gonorrhoea involving isolates with reduced-susceptibility to ceftriaxone.

(250mg and 500mg doses)

Australian wild-type strains previously: Isolates now generally: Australian strains with reduced susceptibility:

< 0.001 mg/L 0.008 mg/L 0.016 mg/L 0.03 mg/L 0.06 mg/L 0.125 mg/L

Japanese H041 strain 2.0 mg/L French F89 strain 1.0 – 2.0 mg/L

Ceftriaxone-resistant NG – Increasing ceftriaxone MICs over time:

Ceftriaxone-resistant NG –

Susceptibilities of the H041 and F89 strains to various antimicrobials:

Antimicrobial H041 F89 Penicillin R R Cefixime R R Ceftriaxone R R Ciprofloxacin R R Azithromycin R R Tetracycline R R

Ceftriaxone-resistant NG –

How were the H041 and F89 treated?

H041 • January 2009, Kyoto, Japan. • Throat sample from a 31-year-old female commercial sex worker. • Initially failed 1 gram ceftriaxone (IM). • Further ceftriaxone treatment was prescribed. However, a culture for test of cure

was not conducted. • A negative result was obtained in April 2009. Spontaneous clearance?

F89 • June 2010, Quimper, France. • Urethral specimen from a 50-year-old man who had sex with men (MSM), • Initially failed 200 mg cefixime (two doses, 6 hours apart) • Successfully treated with gentamicin at 160 mg (IM), single dose. • Verified by culture negativity 3 weeks later.

NG resistance to ceftriaxone: HOW? Chromosomal mutations: • Altered outer membrane protein (porB1b; G120K, A121D) • Increased efflux (mtrR promoter, adenine deletion) • Altered penicillin binding 2 (PBP2) protein

Genetics of chromosomally mediated intermediate resistance to ceftriaxone and cefixime in Neisseria gonorrhoeae. Zhao S, Duncan M, Tomberg J, Davies C, Unemo M, Nicholas RA. Antimicrob Agents Chemother. 2009 Sep;53(9):3744-51.

Outer membrane

cytoplasmic membrane

Peptidoglycan

reduced binding

increased efflux reduced entry

Porin B protein - encoded by porB gene

Penicillin binding protein 2 - encoded by penA gene MtrC-MtrD-MtrE efflux pump

- encoded by mtrCDE operon - regulated by mtrR

NG resistance to ceftriaxone: HOW? • Altered penicillin binding 2 (PBP2) protein

Mosaic PBP2; arising from recombination events.

H041

N. gonorrhoeae wild-type PBP2

N. cinerea PBP2

N. perflava PBP2

N. gonorrhoeae mosaic PBP2

(reduced susceptibility)

1 200 400 600 Amino acids

A501P N. flavescens PBP2

F89

Ceftriaxone-resistant H041 Ceftriaxone-resistant F89

Where are the H041 and F89 strains?

H041 – • Jan 2009 - Japan. • Not seen since. • Questions over fitness?

F89 – • June 2010 - France • May 2011 – Spain (Cámara J et al. JAC2012) • Therefore: Confirmed internal spread, but no further reports.

NOTE: N. gonorrhoeae with mosaic-PBP2 spreading worldwide.

Where are the H041 and F89 strains?

Ciprofloxacin resistance in the USA - 1990–2007

(http://www.cdc.gov)

Are w e here?

So what is being done?

So what is being done? Key components of WHO and CDC action plans: • advocacy for increased awareness on correct use of antibiotics. • effective drug regulations and prescription policies • effective prevention, diagnosis and control of gonococcal infections. • developing procedures for systematic monitoring of treatment failures. • strengthened AMR surveillance • capacity building to establish regional networks of laboratories. • research into alternative effective treatment regimens. • research into newer molecular methods for detecting AMR

So what is being done? New treatments? • Dual therapy: ceftriaxone 500mg plus azithromycin 1g or 2g

Now adopted in the USA, UK and elsewhere. implemented as a strategy to stem AMR development.

• Other suggestions/trials:

Gentamicin Spectinomycin Ertapenem Solithromycin Gemifloxacin

So what is being done? New treatments?

So what is being done? Surveillance: Australian Gonococcal Surveillence Program (AGSP) via A/Prof Monica Lahra & the National Neisseria Network (NNN): • Set up by the late Prof John Tapsall, Prince of Wales Hospital. • Commonwealth funding. • Since 1981, collaborative network of reference labs in each state and territory. • obtains isolates from as wide a section of the community as possible • Considered a flagship for AMR surveillance

Current limitations: • Difficulties obtaining isolates from remote settings. • Increasing use of NAATs for diagnosis.

The Gonorrhoea Resistance Assessment via Nucleic acid Detection (GRAND) Project, Australia.

(funded by the National Health and Medical Research Council)

So what is being done? Molecular methods for detecting AMR?

GRAND project, Australia: Implementing molecular N.gonorrhoeae AMR testing.

Phase 1: To establish a N.gonorrhoeae AMR database. • Isolates (n = 2400) from year 2012 characterised by mutation profiling:

14 SNPs (house keeping genes) to predict MLST type.(Whiley et al.JAC,2013;68(2):322-8.)

15 resistance mutations. • Mutations profiles are being correlated with MIC data, geographical location etc...

Sequenom Massarray iPLEX MALDI-TOF SNP typing platform.

Cost per isolate = approx. $15.0 per isolate

GRAND project, Australia: Implementing molecular N.gonorrhoeae AMR testing.

Phase 1: To establish a N.gonorrhoeae AMR database. • Isolates (n = 2400) from year 2012 characterised by mutation profiling:

14 SNPs (house keeping genes) to predict MLST type.(Whiley et al.JAC,2013;68(2):322-8.)

15 resistance mutations. • Mutations profiles are being correlated with MIC data, geographical location etc...

Phase 2: Application (beginning mid 2013) • Molecular AMR testing of NG-positive samples

Initially from remote settings. Then elsewhere....

What is driving NG resistance?...

Lots of commensal Neisseria species

(including resistant strains)

Pharyngeal NG infection common

Greater potential for sub-lethal antibiotic concentrations, driving:

- selection - recombination

The pharnyx

http://aeremita.deviantart.com/art/Stormy-Sky-3-19004086

What is driving NG resistance?

The rise and spread of ceftriaxone-resistant (XDR) N. gonorrhoeae appears

inevitable & will have severe public health implications. Urgent action is needed at international and local levels to combat this

problem, including: New drugs Improved AMR surveillance.

But also: investigating ways at better controlling gonorrhoea.

improve access to health care, case finding, etc. (particularly in remote Australia).

Conclusions:

Acknowledgements: GRAND study investigators and collaborators, include:

Kirby Institute, UNSW A/Prof Rebecca Guy Prof John Kaldor, Prof Basil Donovan Dr Handan Wand Dr David Regan Baker IDI, NT James Ward Melbourne Sexual Health Centre, Vict. Prof Christopher Fairley A/Prof Marcus Chen & members of the National Neisseria Network, Australia.

Prince of Wales Hospital, NSW A/Prof Monica Lahra Athena Limnios Dr Tiffany Hogan QPID laboratory, QCMRI, Qld A/Prof Theo Sloots A/Prof Michael Nissen Dr Namraj Goire Ella Trembizski Dr Kevin Jacob

Pathology Queensland Prof Graeme Nimmo Dr Cheryl Bletchly Fleur Francis