1. Increasing Rate of Azithromycin Resistance in Treponema pallidum Infections — San Francisco, 2005–2006
Kenneth A. Katz, MD, MSc, MSCE
Lieutenant Commander, U.S. Public Health Service
Epidemic Intelligence Service, Centers for Disease Control and Prevention
STD Prevention and Control Services
San Francisco Department of Public Health
Good morning.

2. Syphilis Sexually transmitted disease Recommended treatment
Caused by Treponema pallidum subsp. pallidum (T. pallidum)
Recommended treatment
Penicillin G benzathine
2.4 million units, intramuscular
Other treatments
Tetracyclines, macrolides, cephalosporins
Penicillin-allergic patients (except pregnant women)
Syphilis is a sexually transmitted disease caused by the bacterium Treponema pallidum subspecies pallidum, or T. pallidum.
The treatment recommended by the Centers for Disease Control and Prevention for most types of syphilis is penicillin G benzathine, as a single intramuscular dose of 2.4 million units for adults.
Other treatment options for early syphilis include tetracyclines, macrolides, and cephalosporins. According to CDC guidelines, those alternative antibiotics may be used to in penicillin-allergic patients, except for pregnant women, who should be desensitized and then treated with penicillin.

3. Incidence of primary and secondary syphilis — San Francisco, 1998–2007
This graph shows the incidence of primary and secondary syphilis in San Francisco between 1998 and 2007, with the year on the x axis and the number of cases per 100,000 residents on the y axis. The incidence rose nearly four-fold between 1999 and This increase resulted from increased numbers of cases among men who have sex with men, who accounted for 20% of primary and secondary syphilis cases in 1998 and over 90% of cases from 2002 through the present.

4. Incidence of primary and secondary syphilis — San Francisco, 1998–2007
Azithromycin (1 g) for sex partners of syphilis patients
In addition to other prevention and control measures, in July 2002 the San Francisco Department of Public Health, or DPH, initiated a program in which sex partners of syphilis patients were treated with a single 1 g dose of azithromycin.

5. Incidence of primary and secondary syphilis — San Francisco, 1998–2007
Azithromycin (1 g) for sex partners of syphilis patients
Azithromycin (2 g) for penicillin-allergic patients
DPH also continued to use azithromycin, in a a 2 g single dose, to treat penicillin-allergic syphilis patients.

6. Treatment failures and resistance
Azithromycin treatment failures, 2002–2003
A2058G mutation
Identified by gene sequencing
Prevents binding of azithromycin to ribosome
Polymerase chain reaction (PCR)-based assay developed
Shortly thereafter, DPH began noting treatment failures in azithromycin-treated patients with primary or incubating syphilis.
The A2058G mutation that confers azithromycin resistance in T. pallidum was identified by gene sequencing of specimens of T. pallidum obtained from two of these patients. That A2058G mutation is an A to G mutation at position 2058 of the 23s ribosomal RNA gene. It confers azithromy resistance by preventing binding of azithromycin to the bacterial ribosome, which azithromycin interferes with protein synthesis to cause a bacteriostatic effect. A polymerase chain reaction-based assay was then developed to identify this mutation.

7. Azithromycin resistance in T. pallidum
San Francisco
1999–2002: 4%
2003: 38%
2004: 54%
Associated with prior azithromycin use
Cessation of azithromycin for syphilis treatment citywide
Also identified in specimens from Seattle, Baltimore, Canada, and Ireland
The assay previously described was used to test specimens of T. pallidum collected from patients seen at the San Francisco City Clinic, the city’s municipal STD clinic, from 1999 to The percentage of azithromycin resistant T. pallidum specimens increased from 4% between 1999 and 2002 to 38% in 2003 and 54% in A case-control study found that resistance was associated with azithromycin use for a nonsyphilis related reason within 30 days prior to syphilis diagnosis. As a result, in September 2004 DPH ceased using or recommending azithromycin for syphilis treatment citywide. Azithromycin resistance has also been identified in T pallidum specimens taken from patients in Seattle, Baltimore, Canada, and Ireland.

8. Study objectives
Assess azithromycin resistance in T. pallidum specimens in 2005 and 2006
Compare resistance rates in San Francisco in 2005 and 2006 to rates from 1999 to 2004
Identify factors associated with presentation with a resistant T. pallidum infection in 2005 and 2006
In order to continue to monitor azithromycin resistance in T. pallidum in syphilis patients in San Francisco, we conducted a study. The objectives of this study were threefold.
First, to assess azithromycin resistance in T pallidum specimens obtained from syphilis patients in San Francisco in 2005 and 2006
Second, to compare resistance rates in 2005 and 2006 to resistance rates from 1999 to 2004;
And third, to identify factors associated with presentation with a resistant form Treponema pallidum in San Francisco since 2004.

9. Study methods Study population Patient data from public health records
Patients with darkfield-positive primary syphilis lesions or moist secondary syphilis lesions
San Francisco City Clinic, 2005–2006
Patient data from public health records
Swabs of lesions from clinical encounters
Azithromycin resistance assay
Comparison data from previous study
Statistical analyses
We studied patients with darkfield-positive primary syphilis lesions or moist secondary syphilis lesions seen at the San Francisco City Clinic, San Francisco’s municipal STD clinic, in 2005 or 2006.
We obtained patient data from public health records.
We obtained Treponema pallidum specimens by swabbing lesions during the clinical encounter.
Testing for azithromycin resistance was done using the polymerase chain reaction-based assay mentioned previously to detect the A2058G mtutation.
Comparison data for years prior to 2005 were taken from a previous study of San Francisco City Clinic patients that used these same methods.
Statistical analyses included Wilcoxon rank sum tests to compare continuous variables, Fisher’s exact tests to compare categorical variables, and logistic regression analyses to assess trends in azithromycin resistance by year.

10. Specimens analyzed Primary syphilis: swabs from 44 patients
Secondary syphilis: swabs from 3 patients
Syphilis, undocumented stage: swabs from 2 patients
Swabs from 49 patients sent for analysis
Swabs of lesions were obtained from 44 patients with primary syphilis.
Swabs of lesions were also obtained from 3 patients with secondary syphilis and 2 patients with syphilis of undocumented stage.
Of those 49 swabs that were sent for azithromycin resistance analysis, 39 contained sufficient DNA for analysis, including 37 from patients with primary syphilis and 2 from patients with secondary syphilis. Of the 39 analyzed swabs, 17 were obtained in 2005 and 22 in 2006.
Swabs from 39 patients with sufficient T. pallidum DNA for analysis; 37 primary, 2 secondary

11. Patient characteristics (N=39)
Median age in years: 45 (range, 23–62)
Male: 97%
Men who have sex with men: 92%
HIV-infected: 41%
Azithromycin use within three months: 3%
Of the 39 patients studied, the median age was 45, with a range of 23 to percent of patients were men, 92 percent were gay men or other men who have sex with men, and 41% were co-infected with HIV.
Comparing the 37 patients with primary syphilis whose specimens were analyzed to all primary syphilis patients seen at the City Clinic in 2005 and 2006, the distribution of these characteristics did not differ significantly.

12. Azithromycin resistance in T
Azithromycin resistance in T. pallidum specimens — San Francisco, 1999–2006
76.5%
77.3%
This graph shows, on the x axis, the year or years studied, and, on the y axis, the percentage of T. pallidum specimens resistant to azithromycin. Comparison data from 1999 to 2004 are shaded gray, while those from the current study are in turquoise to 2002 are combined here because there were only 24 specimens tested during this period, resulting in unstable yearly estimates. In 2005 and 2006, the percentage of azithromycin resistant specimens were 76.5% and 77.3%, respectively.

13. Azithromycin resistance in T
Azithromycin resistance in T. pallidum specimens — San Francisco, 1999–2006
76.5%
77.3%
P < 0.001
Those proportions represent a statistically significant continued increase in azithromycin resistance since None of the patients seen after 2004 were treated for syphilis with azithromycin.

14. Patient characteristics by azithromycin-resistance status
Sensitive (N=9)
Resistant (N=30)
P value
Median age in years 47
43.5
0.28
Male sex (%)
100 97
0.77
Gay men or other MSM (%) 89 93
0.80
HIV-infected (%) 23 43
0.55
Azithromycin use within three months prior to diagnosis (%) 3
Characteristics of patients, including age, sex, sexual orientation, hiv infection status, and azithromycin use within three months prior to diagnosis, did not significantly differ between those with sensitive and resistant T pallidum specimens.

15. Limitations Subset of patients
Only 25% of primary and secondary syphilis patients in San Francisco during study period seen at City Clinic
Limited power to detect differences in patient characteristics
Only 39 patients studied
This investigation was subject to at least two limitations. First, we studied only a subset of syphilis patients. Of all patients with primary or secondary syphilis in San Francisco during the study period, only 25% were seen at San Francisco City Clinic.
Second, we had limited power to detect associations between patient characteristics, including HIV status, and azithromycin resistance, with only 39 patients studied.
************
Of those, patients with darkfield-positive lesions comprised only 64% of primary syphilis patients and 14% of secondary syphilis patients.
Of patients with darkfield-positive lesions seen at City Clinic, 84% of primary syphilis patients and 66% of secondary syphilis patients had azithromycin resistance status of T. pallidum infections analyzed. In sum, we were able to analyze resistance status for only 54% of primary syphilis infections and less than 1% of secondary syphilis infections seen at City Clinic. We compared characteristics of patients with primary syphilis studied with primary syphilis patients in San Francisco not studied. The one characteristic that significantly differed was HIV status. Those who were studied were 43% less likely to be HIV positive than those who were not studied. Although there is no evidence that azithromycin resistance status is associated with HIV status, this lack of association is based on a limited number of subjects.

16. Summary Azithromycin resistance
2005: 76.5%
2006: 77.3%
Continued increase in resistance since 1999
Despite decreased use of azithromycin for syphilis
No patient characteristics associated with resistant infection in 2005–2006
Age, sex, sexual orientation, HIV-infection status, recent azithromycin use
In summary, azithromycin resistance was present in 76.5% of T. pallidum specimens in 2005 and 77.3% of specimens in 2006.
Those percentages represent a continued statistically significant increase in azithromycin since 1999, despite decreased use of azithromycin for syphilis.
No patient characteristics were associated with presentation with a resistant infection in 2005 and 2006, including age, sex, sexual orientation, HIV-infection status, and recent azithromycin use.

17. Recommendations Vigilance for treatment failures
Developing countries
Drug development for syphilis
Surveillance for azithromycin resistance
CDC recently launched nationwide surveillance project to assess azithromycin resistance in T. pallidum
Based on the findings of this investigation, there is, first, a need for vigilance for treatment failures in syphilis patients treated with azithromycin. This is particularly the case for developing countries, where azithromycin is currently being studied as a potential first-line treatment for syphilis because it can be given orally, avoiding the need for injection equipment and trained medical personnel to administer injection.
Second, there is a continued need for development of new antibiotics to treat syphilis.
Third, there is also a need for surveillance for azithromycin resistance in Treponema pallidum in areas where azithromycin may be used to treat syphilis. To this end, the CDC this year launched a nationwide surveillance project to assess azithromycin resistance in T. pallidum.

18. Real-time PCR assay Uses melting curve to detect mutation (Sensitive)
(Resistant)
Of note, DPH and collaborators have recently developed an enhanced real-time PCR assay for azithromycin resistance that can generate results in less than four hours, compared to more than 10 hours required by the other assay. This new assay was 100% sensitive and specific for detecting the A2058G mutation compared to the other assay. This new assay uses a melting curve analysis to determine the presence of the A2058G mutation. This graph shows temperature on the x axis and fluorescence, corresponding to melting peaks, on the y axis. The curves in the figure represent the different melting curves for an azithromycin sensitive T. pallidum strain, the Nichols strain, and an azithromycin resistant T pallidum strain that contains the A2058G mutation, the Street 14 strain. This ability to more rapidly assess T. pallidum specimens holds promise for clinical as well as epidemiologic use.
Pandori M, et al. Detection of azithromycin resistance in Treponema pallidum by real-time PCR.. Antimicrob Agents Chemother. 2007;51:

19. Acknowledgments San Francisco Department of Public Health
Jeff Klausner
Katherine Ahrens
Kyle Bernstein
Joseph Engelman
Susan Philip
Mark Pandori
University of Washington
Sheila Lukehart
Charmie Godornes
Centers for Disease Control and Prevention
Sheryl Lyss
Edward Weiss
University of California at Berkeley
Keith Hermanstyne
In closing, I would like to acknowledge the people shown here for their help in completing this project. I would be happy to take any questions. Thank you.
The findings and conclusions in this report are those of the author and do not necessarily represent the views of the Centers for Disease Control and Prevention.

20. Additional slides

21. Detection of A2058G mutation
PCR
Sensitive
Restriction enzyme digestion
Sensitive – single band
This slide illustrates the PCR based technique that was used to detect the A2058G mutation. Briefly, after a syphilis lesion is swabbed, (1) the portion of DNA containing the 23s ribosomal RNA gene is amplified using PCR. (2) The DNA is then subjected to digestion by a restriction enzyme. In a T pallidum specimen sensitive to azithromycin, as shown here, the restriction enzyme used does not cleave the strand of DNA that contains the 23s rRNA gene. (3) Therefore, when the DNA is run across an agarose gel using electrophoresis, only one large band, representing uncleaved DNA, is present. In the gel shown here, the columns with only one large band at the top represent specimens that lack the A2058G mutation and therefore are sensitive to azithromycin.
Agarose gel

22. Detection of A2058G mutation
PCR
Resistant
Restriction enzyme digestion
Resistant – two bands
In the case of azithromycin resistant specimens, however, the restriction enzyme does cleave the DNA into two pieces. Therefore, when DNA from resistant specimens are run across an agarose gel, two bands are seen. In the gel pictured here, the columns that show two distinct bands, one at the top and one at the bottom, are from specimens of T pallidum that are resistant to azithromycin.
Agarose gel

23. Limitations Subset of patients Patients seen at City Clinic (25%)
Patients with primary or secondary syphilis in San Francisco
This investigation was subject to at least two limitations. First, we studied only a subset of syphilis patients. Of all patients with primary or secondary syphilis in San Francisco during the study period, only 25% were seen at San Francisco City Clinic.

24. Limitations Subset of patients
Patients with darkfield-positive lesions (primary, 64%; secondary, 14%)
Patients seen at City Clinic (25%)
Patients with primary or secondary syphilis in San Francisco
Of those, patients with darkfield-positive lesions comprised only 64% of primary syphilis patients and 14% of secondary syphilis patients.

25. Limitations Subset of patients
Patients with darkfield-positive lesions (primary, 64%; secondary, <1%)
Patients seen at City Clinic (25%)
Patients with primary or secondary syphilis in San Francisco
Patients with swabs analyzed (primary, 84%; secondary, 66%)
Of patients with darkfield-positive lesions seen at City Clinic, 84% of primary syphilis patients and 66% of secondary syphilis patients had azithromycin resistance status of T. pallidum infections analyzed. In sum, we were able to analyze resistance status for only 54% of primary syphilis infections and less than 1% of secondary syphilis infections seen at City Clinic. We compared characteristics of patients with primary syphilis studied with primary syphilis patients in San Francisco not studied. The one characteristic that significantly differed was HIV status. Those who were studied were 43% less likely to be HIV positive than those who were not studied. Although there is no evidence that azithromycin resistance status is associated with HIV status, this lack of association is based on a limited number of subjects.

26. Limitations Subset of patients
Limited power to detect differences in patient characteristics (n=39)
That leads to the second limitation: we had limited power to detect associations between patient characteristics, including HIV status, and azithromycin resistance, with only 39 patients in the sample.