1. Azithromycin resistance in Treponema pallidum in the United States and Ireland is associated with a mutation in the 23S rRNA gene University of Washington San Francisco Department of Health St. James Hospital, Dublin, Ireland Johns Hopkins University

2. Major points Observations of clinical failure Searching for a resistance-associated mutation Functional resistance in vivo—rabbit model Development of a rapid screening method Survey of samples from different geographical regions

3. Benzathine penicillin G Depot form of penicillin Long half-life Single dose for early syphilis Painful intramuscular injection

4. Azithromycin as an Alternative Therapy Macrolide family Long tissue half-life Oral administration

5. Published Studies of Azithromycin Treatment for Syphilis ReferenceStage of syphilisDose Lukehart et al, 1990Primary syphilis (rabbit model) Equiv of 500 mg, 1 g, or 2 g/day, daily X 15 d Verdon et al, 1994 1 , 2  500 mg daily X 10 d Mashkilleyson et al, 1996 “Seropositive” syphilis 500 mg daily X 10 d; or 500 mg alternate days X 11 d Hook et al, 1999Incubating1 g single dose Gruber et al, 2000Early syphilis1 g on first day, then 500 mg/day X 8 d Hook et al, 2002Early syphilis2 g single dose, or 2 g weekly X 2 wks

6. Case Report – July, 2003 Day 0: 33 yo HIV-infected man presented to ER with nontender penile ulcer x 1 week –Rx 2 g azithromycin PO Day 1: Took additional 1 g azithromycin PO Day 2: Presented SF STD Clinic for f/u –1 cm indurated, nontender ulcer on glans penis –Left inguinal adenopathy –Serum VDRL NR; DF Neg

7. Case Report – July, 2003 Day 5:RTC with persistent ulcer –DF pos for T. pallidum –TP-PA Reactive –Rx 2.4 MU BPG Day 19: RTC – resolution of lesion 3 mo: VDRL Nonreactive

8. Sequence Analysis of 23S rRNA Gene in Selected T. pallidum Strains T. pallidum strain 23S rRNA geneMutation NicholsTAGACGGAAAGACCCCWild type Street 14TAGACGGGAAGACCCC AGAG CA 42TAGACGGGAAGACCCC AGAG

9. Functional Evidence of Azithromycin Resistance – Rabbit Model Intradermal infection 10 6 Tp/site –Nichols or Street 14 strain Treatment groups –Untreated –BPG –Erythromycin (2 g/day x 14 d) –Azithromycin (1 g/day x 14d)

10. Darkfield Positive after Therapy NicholsStreet 14 Untreated 3/3 BPG 0/3 Erythromycin 0/33/3 Azithromycin 0/33/3 Outcome – Rabbit Study

11. Rapid screening method PCR amplify 23S rRNA gene fragment 628 bp TAGACGGAAAGACCCC TAGACGGGAAGACCCC Mbo II Cut with Mbo IINo Mutation Yes 440 bp188 bp 628 bp

12. 23S rRNA Gene Mutation in Treponema pallidum Samples Geographical Location Dates of sample collection Number of samples with mutation/Total amplifiable samples (%) San Francisco, CA 1999-20021/25 ( 4%) 20039/31 (29%) Seattle, WA2001-20033/23 (13%) Baltimore, MD1998-20002/19 (11%) Dublin, Ireland200215/17 (88%) Historical strains from multiple locations 1912-19871/18 ( 6%)

13. Restriction Digestion of 23S rRNA Gene Amplicon

14. Sequence Analysis of 23S rRNA Gene in Selected T. pallidum Strains T. pallidum strain 23S rRNA geneMutation NicholsTAGACGGAAAGACCCCWild type Street 14TAGACGGGAAGACCCC AGAG CA 42TAGACGGGAAGACCCC AGAG CA 61TAGACGGGAAGACCCC AGAG Dub 21TAGACGGGAAGACCCC AGAG Dub25TAGACGGGAAGACCCC AGAG Dub 58TAGACGGGAAGACCCC AGAG Dub 49TAGACGGGAAGACCCC AGAG UW 133TAGACGGGAAGACCCC AGAG UW 157TAGACGGGAAGACCCC AGAG

15. Summary A → G mutation in 23S rRNA gene confers azithromycin resistance in T. pallidum Rapid restriction digestion method can detect mutation in swab specimens from ulcers Mutation is found in multiple geographical locations

16. Implications The true prevalence of resistant strains is unknown, but worrisome. If azithromycin is used for treatment of syphilis, close follow-up must be assured. In geographical regions where resistance may be relatively high, the recommended treatment for syphilis should remain penicillin.

17. Authors University of Washington –Sheila Lukehart, Ph.D. –Christina Marra, M.D. –Charmie Godornes –Barbara Molini, M.S. –Patricia Sonnet St. James Hospital, Dublin, Ireland –Susan Hopkins, M.D. –Fiona Mulcahy, M.D. San Francisco Department of Health –Joseph Engelman, M.D. –Samuel J. Mitchell, M.D., Ph.D. –Jeffrey D. Klausner, M.D., M.P.H. Johns Hopkins University –Anne Rompalo, M.D.