It was first reported in 1943 that penicillin cured syphilis,1 Benzathine penicillin G, introduced in 1956,2 has been used in the treatment of syphilis ever since. Despite its long-term and widespread use, there has been no reported resistance of Treponema pallidum to penicillin, and parenteral penicillin remains the treatment of choice for syphilis.3,4 Azithromycin has been investigated as alternative syphilis therapy5–8; it is orally administered, has a long half-life, and is used in the treatment of other sexually transmitted infections.9 It is an attractive as an epidemiologic treatment of syphilis contacts.
In a multicenter study in 2004, Lukehart et al10 reported on azithromycin resistance in the United States and Ireland; 28% of isolates were found to have the A2058G mutation. The center with the highest proportion of azithromycin-resistant samples was Dublin, where 15/17 (88%) were found to have the mutation compared with 22% in San Francisco, 13% in Seattle, and 11% in Baltimore. In San Francisco, the proportion of samples containing the mutation was also found to have increased from 4% in the period 1999 to 2002 to 37% in 2003.10 It is interesting to note that when eighteen historical samples were analyzed from various locations from 1912 to 1987, only one, the street 14 strain, was found to have macrolide resistance.
Several centers have since evaluated the rate of azithromycin resistance among patients presenting with syphilis. In San Francisco, between 2000 and 2004, 46 of a total of 118 contained mutant 23S ribosomal RNA genes.11 The mutation was identified in 1/25 (4%) isolates from 2000 to 2002 and in 13/32 (41%) from 2003. This prompted the discontinuation of the use of azithromycin for the treatment of primary and secondary syphilis and for incubating syphilis in San Francisco. A similar pattern of emerging resistance was seen in British Columbia, Canada,12 and in Seattle; the odds of a syphilis strain having the mutation increased significantly during the years 2001 to 2005 (P = 0.006).13
More recently, worldwide reported rates of azithromycin resistance vary, with no resistant isolates identified in Madagascar (2008),14 28.6% in Alberta, Canada (2007–2008),15 and 100% in Shanghai, China (2007–2008).16
As an increase in the number of new syphilis diagnoses became apparent,17 the opportunity arose to investigate the current rates of T. pallidum azithromycin resistance in Dublin.
Samples were collected from patients attending the Genitourinary Medicine and Infectious Diseases (GUIDE) clinic, St James’s Hospital, Dublin, from January 2009 to January 2010. Exudate from patients with lesions, which clinically resembled primary syphilis, was analyzed using dark ground microscopy. The remaining exudate was transferred to an Abbott multi-Collect Specimen Collection kit containing 250 µL of transport buffer (Multi-collect Buffer; Abbott Molecular, Des Plaines, IL). EDTA whole blood was drawn from patients who presented with potential incubating syphilis or early infectious syphilis (i.e., clinical primary or secondary disease or those asymptomatic with positive serologic test result who acquired their syphilis within the last 2 years). Cerebrospinal fluid (CSF) was collected from patients undergoing lumbar puncture for investigation of neurosyphilis and tissue from patients who underwent biopsy for diagnostic purposes (2 skin biopsies, 1 vitreal biopsy). The ethics committee of St. James’s Hospital approved the study.
Specimens were stored at −20°C and polymerase chain reaction (PCR) analysis was performed within 2 months. DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Crawley, United Kingdom) as per the manufacturer’s instructions. The PCR assay used to detect T. pallidum has been previously published by Leslie et al.18 Briefly, the T. pallidum target was the 67–base pair (bp) sequence within the polA gene, sequence (nucleotides 2001 to 2067) (GenBank accession no. TPU57757). Primer/probe Sequence (5′-3′) TP-F-AGG ATC GCC CAT ATG TCC AA′, TP-R-GTC AGC GTC TCA TCA TTC CAA′, TP-MGB- FAM- ATG CAC CAG CTT CGA-NFQ. The amplification process consisted of incubation 20 seconds at 95°C (AmpliTaq Gold enzyme activation), 45 cycles of 3 seconds at 95°C (denaturation), and 30 seconds at 60°C (combined annealing and primer extension) using an ABI Prism 7500 Fast sequence detection system (Applied Biosystems). Negative (Normal human serum, Sigma-Aldrich, Ireland) and nontemplate controls were included in each run.
Real-time results are expressed as cycle threshold (Ct) values. The Ct value is generated as the amplified sample crosses the threshold level of detection set during the exponential phase of the PCR reaction; as a result lower Ct values will be seen with larger amounts of DNA present in a sample.
To determine azithromycin resistance, all samples were tested using a nested PCR as previously described by Lukehart et al10 and Pandori et al.19 The outer primers (TP-23S-OF- GTA CCG CAA ACC GAC ACA G′, TP-23S-OR-TAG GTG GGC GGT TTG ACT) targeted a 628-bp sequence; the inner primers (TPAZ-F -GAC TCT GGA CAC TGT CTC G, TPAZ-R-CAA CAG TGA AAT ACC ACC C) target was a 201-bp sequence. In addition to extracted DNA from clinical samples, Nicholls strain DNA, street 14 strain DNA, and 3 control samples known to be azithromycin resistant were included (compliments of Lukehart, Seattle, WA). Also included in each run were a nontemplate control and a negative control. The PCR cycling parameters were 3 minutes at 94°C (initial inactivation), followed by 35 cycles of 60 seconds at 94°C (denaturation), 1 minute at 55°C (annealing), and extension 1 minute at 72°C. Final extension step was 10 minutes at 72°C.
DNA sequencing was performed using the BigDye terminator V3.1 cycle sequencing kit (Applied Biosystems, Warrington, United Kingdom), as per manufacturer’s instructions using the ABI Prism 3130xl DNA analyzer (Applied Biosystems). Sequence analysis was carried out using DNASTAR Lasergene (DNASTAR, Madison, WI). A consensus sequence was assembled and edited using SeqMan sequence analysis software. All nucleotide were aligned using MegAlign, and the A2058G sequence mutation was identified.
A total of 107 samples from 92 patients were collected. The mean age of the patients was 37.8 years (range, 19–64 years). Of the 92 patients, 88 (95.7%) were male. The largest risk group was men who have sex with men (72/92 [78.3%]). The samples analyzed and results are presented in Table 1.
The mean Ct of the blood samples analyzed was 35.76 (95% confidence interval, 34.1–37.41); this value was significantly higher than the mean Ct value for the genital exudate samples at 28.44 (95% confidence interval, 26.26–30.62; P < 0.0001).
Of the 21 genital exudate samples, all 21 patients had a dark ground microscopy sample performed on the same day. There were 2 discrepant results; 2 samples were dark ground microscopy negative and PCR positive. Of the 2 samples with a negative dark ground microscopy result, 1 had relatively high Ct value on PCR of 38, whereas the other had a Ct value of 32.54. Three patients had dark ground microscopy result, which was positive; however, their PCR result was negative. Compared with dark ground microscopy, the sensitivity and specificity were 86.3% and 75%, respectively. The κ value of 0.614 was found for agreement between the tests.
Of the 30 patients with positive PCR results, 29/30 (96.7%) had positive syphilis serologic test result; results were unavailable for 1 patient. Of the patients with negative PCR results, 44/62 (70.97%) had positive serologic test result.
Samples from 30 patients were analyzed for the presence of the A2058G mutation. A total of 12 of blood samples, 19 genital exudates, 1 CSF, and 2 tissue samples were analyzed. Of the 30 samples analyzed, 29 samples successfully sequenced. The remaining sample failed to amplify DNA product. Of the 29 samples that successfully sequenced, 27 (93.1%) were found to contain the A2058G mutation. It is interesting to note that the 2 patients who had isolates, which did not contain the A2058G mutation, both described the risks for acquisition of their syphilis to be outside Ireland.
In 2004, when syphilis treatment failures after azithromycin therapy were reported20 and the A2058G mutation associated with azithromycin resistance was described,21 Dublin had the highest rate of azithromycin resistance.10 It seems little has changed; 27/29 (93.1%) of isolates are azithromycin resistant. In the 2 patients with sensitive isolates, the risk for acquisition of their syphilis was outside Ireland, suggesting that perhaps the situation may well be worse than reported in 2004.
Marra et al13 have shown that the mutation is present in greater than 2 syphilis strains, which refutes the argument of a single resistant strain circulating among some high-risk populations. In addition, they found that patients who had received azithromycin therapy in the 12 months previously were twice as likely to have a resistant strain of syphilis, suggesting that antibiotic selection has contributed to the increase in macrolide resistant T. pallidum. Azithromycin is commonly used in Ireland for the treatment of sexually transmitted infections such as Chlamydia trachomatis and nonspecific urethritis.
Although the findings presented are specific to Dublin, with increased globalization practitioners need to be aware of potential resistance patterns in patients presenting after travel or migration. Any plans to establish Irish guidelines for the management and treatment of syphilis must take into consideration the high level of macrolide resistance in T. pallidum isolates in Dublin. Little has changed since the original study in 2004. Azithromycin cannot be recommended for use in the treatment of syphilis in Dublin and should not be used in the epidemiologic treatment of patients with syphilis contacts.
1. Mahoney JF, Arnold RC, Harris A. Penicillin treatment of early syphilis—A preliminary report. Am J Public Health Nations Health 1943; 33: 1387–1391.
2. Smith CA, Kamp M, Olansky S, et al.. Benzathine penicillin G in the treatment of syphilis. Bull World Health Organ 1956; 15: 1087–1096.
3. Workowski KA, Berman S, Centers for Disease Control and Prevention. Sexually transmitted diseases treatment guidelines, 2010. MMWR Recomm Rep 2010; 59: 1–110.
4. Kingston M, French P, Goh B, et al.. UK National Guidelines on the Management of Syphilis 2008. Int J STD AIDS 2008; 19: 729–740.
5. Hook EW 3rd, Martin DH, Stephens J, et al.. A randomized, comparative pilot study of azithromycin versus benzathine penicillin G for treatment of early syphilis. Sex Transm Dis 2002; 29: 486–490.
6. Kiddugavu MG, Kiwanuka N, Wawer MJ, et al.. Effectiveness of syphilis treatment using azithromycin and/or benzathine penicillin in Rakai, Uganda. Sex Transm Dis 2005; 32: 1–6.
7. Lukehart SA, Fohn MJ, Baker-Zander SA. Efficacy of azithromycin for therapy of active syphilis in the rabbit model. J Antimicrob Chemother 1990; 25 (suppl A): 91–99.
8. Verdon MS, Handsfield HH, Johnson RB. Pilot study of azithromycin for treatment of primary and secondary syphilis. Clin Infect Dis 1994; 19: 486–488.
9. Geisler WM. Management of uncomplicated Chlamydia trachomatis
infections in adolescents and adults: Evidence reviewed for the 2006 Centers for Disease Control and Prevention sexually transmitted diseases treatment guidelines. Clin Infect Dis 2007; 44 (suppl 3): S77–S83.
10. Lukehart SA, Godornes C, Molini BJ, et al.. Macrolide resistance in Treponema pallidum
in the United States and Ireland. N Engl J Med 2004; 351: 154–158.
11. Mitchell SJ, Engelman J, Kent CK, et al.. Azithromycin-resistant syphilis infection: San Francisco, California, 2000–2004. Clin Infect Dis 2006; 42: 337–345.
12. Morshed MG, Jones HD. Treponema pallidum
macrolide resistance in BC. CMAJ 2006; 174: 349.
13. Marra CM, Colina AP, Godornes C, et al.. Antibiotic selection may contribute to increases in macrolide-resistant Treponema pallidum
. J Infect Dis 2006; 194: 1771–1773.
14. Van Damme K, Behets F, Ravelomanana N, et al.. Evaluation of azithromycin resistance in Treponema pallidum
specimens from Madagascar. Sex Transm Dis 2009; 36: 775–776.
15. Martin IE, Tsang RS, Sutherland K, et al.. Molecular characterization of syphilis in patients in Canada: Azithromycin resistance and detection of Treponema pallidum
DNA in whole-blood samples versus ulcerative swabs. J Clin Microbiol 2009; 47: 1668–1673.
16. Martin IE, Gu W, Yang Y, et al.. Macrolide resistance and molecular types of Treponema pallidum
causing primary syphilis in Shanghai, China. Clin Infect Dis 2009; 49: 515–521.
17. Muldoon E, Mulcahy F. Syphilis resurgence in Dublin, Ireland. Int J STD AIDS; 22: 493–497.
18. Leslie DEA, Karapanagiotidis F, Leydon T, et al.. Development of a Real-time PCR Assay to detect Treponema pallidum
in clinical specimens and assessment of the assay’s performance by comparison with serological testing. J Clin Microbiol 2006; 45: 93–96.
19. Pandori MW, Gordones C, Castro L, et al.. Detection of azithromycin resistance in Treponema pallidum
by real-time PCR. Antimicrob Agents Chemother 2007; 51: 3425–3430.
20. Centers for Disease Control and Prevention. Azithromycin treatment failures in syphilis infections—San Francisco, California, 2002–2003. MMWR Morb Mortal Wkly Rep 2004; 53: 197–198.
21. Stamm LV, Bergen HL. A point mutation associated with bacterial macrolide resistance is present in both 23S rRNA genes of an erythromycin-resistant Treponema pallidum
clinical isolate. Antimicrob Agents Chemother 2000; 44: 806–807.