P2.082 Post-Treatment Detection of Azithromycin in High-Vaginal Swabs Using Liquid Chromatography and Tandem Mass Spectrometry (LC-MS/MS)

Abstract

Results Of the 156 specimens, 83 N. gonorrhoeae strains were tested for antimicrobial susceptibilities to 18 agents. The prevalence of β-lactamase producing strains and chromosomally-mediated resistant strains were 7.2% and 16.5%, respectively. Against cepharosporins, one strain was resistant to cefixime with MIC 0.5 μg/ml. There was not resistant strain to ceftriaxone, but the 7 strains (8.4%) had MIC 0.125 μg/ml. The MIC of fluoroquinolones to all strains showed a bimodal distribution. The values of MIC90 of ciprofloxacin and levofloxacin were 16 and 8 μg/ml, respectively. Sitafloxacin, one of fluoroquinolones had strong activity to N. gonorrhoeae strains and the value of MIC90 was 0.25 μg/ml. The MIC of azithromycin in 2 strains was 2 μg/ml, but no high-level resistance to macrolides was detected. Conclusion The first national surveillance for antimicrobial susceptibilities of N. gonorrhoeae was performed. Fluoroquinolone-resistance N. gonorrhoeae strains were spread in Japan. The resistant rate of azithromycin resistant was 2.4%. Introduction Recent data have raised questions over the efficacy of azithromycin 1g for the treatment of chlamydia infection. In order to measure effective absorption, we developed a protocol to quantify the concentration of azithromycin using liquid chromatography and tandem mass spectrometry (LC-MS/MS) in self-collected high-vaginal swabs. Methods Ten healthy women were asked to self-collect a highvaginal swab (baseline) prior to taking a 1g dose of azithromycin. A blood sample was collected four hours later to determine plasma concentrations of azithromycin. Participants then self-collected a high vaginal swab each day for a further 9 days. All swabs were preserved in 1ml of 100% Methanol and stored at -80°C prior to analysis. One ml of chloroform containing 10mg/ml of Leucine enkephalin as an internal standard was added to extract azithromycin. Azithromycin concentrations were calculated using a validated LC-MS/MS method. Data were normalised to the internal standard and to membrane lipid concentrations, measured in the same samples using LC-MS/MS. Results Azithromycin was detected at varying concentrations in all 10 women in all post-treatment samples. The highest average normalised azithromycin concentration of 953ng/ml (range = 267-2200ng/ml, standard error of mean (sem) = 181ng/ml) was detected on day 2 post-treatment. The lowest average azithromycin concentration was 164ng/ml (range = 51-387ng/ml, sem = 42ng/ml), 9 days post-treatment. The average concentration of azithromycin detected in blood samples was 339ng/ml (range = 107-628ng/ml, sem = 57ng/ml). In 9/10 women azithromycin concentrations remained above 64ng/ml, the hypothesised mean inhibitory concentration (MIC) of azithromycin for chlamydia, for the entire 9 days. Conclusion We have validated a method for detecting the azithromycin concentration in self-collected high-vaginal samples using LC-MS/MS. Azithromycin concentrations remained above the Post-treatment Detection of azithromycin in high-

A cohort study of Chlamydia trachomatis treatment failure in women: a study protocol

“…Azithromycin levels are thought to remain well above the reported MIC for chlamydia for between 10 to 14 days post-treatment with 1 g dose [30,31]. Azithromycin levels in high-vaginal material (cells and mucus) will be measured using a validated liquid chromatography – tandem mass spectrometry method (LC-MS/MS) [47-50]. An azithromycin standard curve will be prepared in high-vaginal specimens from a separate individual not exposed to azithromycin, and azithromycin concentrations detected by LC-MS/MS will be normalised to lipid concentrations [50].…”

“…Azithromycin levels in high-vaginal material (cells and mucus) will be measured using a validated liquid chromatography – tandem mass spectrometry method (LC-MS/MS) [47-50]. An azithromycin standard curve will be prepared in high-vaginal specimens from a separate individual not exposed to azithromycin, and azithromycin concentrations detected by LC-MS/MS will be normalised to lipid concentrations [50]. Azithromycin concentrations in high-vaginal specimens will be normalised to membrane lipid concentrations and then calculated using the standard curve.…”