Efficacy of Sitafloxacin for Mycoplasma genitalium in an Era of Increasing Antimicrobial Resistance

Abstract Antimicrobial resistance in Mycoplasma genitalium is rising globally and antimicrobial options are limited. We evaluated the efficacy of sitafloxacin regimens for macrolide-resistant M genitalium at Melbourne Sexual Health Centre, Australia, between January 2017 and February 2022. Before June 2017, patients received doxycycline followed by sitafloxacin; subsequently, patients received doxycycline followed by combined doxycycline + sitafloxacin. Of 229 patients treated with a sitafloxacin regimen, 80.6% experienced microbial cure. Sitafloxacin cured 94.2% of infections that had not previously failed moxifloxacin and 69.5% of infections that had; prior failure of moxifloxacin was associated with an 8-fold odds of sitafloxacin failure. There was no difference in cure between sequential monotherapy and combination therapy when patients were stratified by past failure of moxifloxacin (P > .05); however, small numbers limited comparisons. Sitafloxacin was well tolerated and still achieved 70% cure in patients in whom moxifloxacin had failed. These data highlight the benefit of incorporating relevant fluoroquinolone resistance markers into assays to assist clinical decision making.

Mycoplasma genitalium is a sexually transmitted bacterium with an evolving antibiotic resistance profile that impacts our ability to cure infections [1].Mycoplasma genitalium has no cell wall, restricting antibiotic choice to agents that act on protein or DNA synthesis including tetracyclines, streptogramins, macrolides, and later-generation fluoroquinolones.The prevalence of M genitalium in the general population is estimated to be 1%-3%, and macrolide resistance is reported to be >50% in many settings, including Australia [2].Extended-spectrum fluoroquinolones, such as moxifloxacin, are used to treat macrolide-resistant M genitalium.However, some singlenucleotide polymorphisms (SNPs) in the parC gene, such as that conferring the amino acid change S83I, have been shown to increase the minimum inhibitory concentration (MIC) and reduce the efficacy of moxifloxacin [3].We have shown that these SNPs frequently coexist with macrolide resistance mutations, further restricting treatment options [2].
At Melbourne Sexual Health Centre (MSHC), S83I was detected in 23% of macrolide-resistant strains in 2020 [4], and dual-class resistant infection (macrolide and quinolone resistance) increased from 9% in 2012-2013 to 16% in 2016-2018 [4].This rise has significantly impacted the efficacy of moxifloxacin in our service with increasing numbers of patients requiring additional antibiotics.We previously reported a small case series of 12 patients with persistent M genitalium who had failed moxifloxacin, pristinamycin, and minocycline, and found that treatment with combination 100 mg doxycycline and 100 mg sitafloxacin twice daily (BID) for 1 week cured 11 of 12 infections [5].Sitafloxacin is a quinolone that is available across the Asia-Pacific region but is limited elsewhere.In Japan, sitafloxacin has been used for a number of years as monotherapy for M genitalium and was reported to achieve cure of approximately 90% [6,7].However, in Australia, due to concerns about antimicrobial stewardship, sitafloxacin is now reserved for the treatment of resistant M genitalium infections.As sitafloxacin is not registered with the Therapeutic Goods Administration in Australia, it is imported from overseas, making it costly and hence reserved for treating resistant M genitalium infection.To attain greater understanding of the efficacy and use of sitafloxacin in a larger number of patients, and in the context of rising fluoroquinolone resistance in our clinic population, we undertook an evaluation of all cases treated with sitafloxacin over the past 5 years at MSHC.

METHODS
We conducted a retrospective evaluation of patients infected with M genitalium who were treated with a sitafloxacin regimen at MSHC in Melbourne, Australia, from January 2017 to February 2022.MSHC is the only public clinic treating sexually transmitted infections (STIs) in a city of >5 million.From January 2017 to March 2021, M genitalium was tested for using the ResistancePlus MG assay (SpeeDx Pty Ltd, Sydney, Australia).After March 2021, in the final year of the study, the diagnostic assay changed to transcription-mediated amplification (TMA; Aptima MG assay; Hologic Gen-Probe Panther system), with reflex macrolide resistance testing of positives using the ResistancePlus MG assay.
In the first 5 months, from January to May 2017, sitafloxacin was firstline for patients with macrolide-resistant M genitalium and prescribed as sequential monotherapy, which involved doxycycline (100 mg BID) for 7 days, followed by sitafloxacin (100 mg BID) for 7 days.Due to antimicrobial stewardship concerns, from June 2017, sitafloxacin was reserved for macrolide-resistant infections that had failed 1 or more other regimens and was used predominantly as sequential combination therapy [8]: doxycycline (100 mg BID) for 7 days followed immediately by combination therapy with doxycycline and sitafloxacin both 100 mg BID for a further 7 days.All patients were asked to return for a test of cure (TOC) 14-21 days after completing sitafloxacin.Clinicians performing a TOC recorded information into an electronic template that captured evolution of genital symptoms after treatment, medication adherence, and side effects, as well as sex since treatment and partner testing and treatment status, to ascertain the risk of reinfection.
Patients were included in the study if they had a TOC performed within 14-90 days of completing sitafloxacin.Patients were excluded if they took less than half of the sitafloxacin doses or had a high risk of reinfection.Consistent with prior studies [4,8,9], high risk of reinfection was defined as self-report of condomless sexual intercourse after treatment with a regular partner who had not been tested and/or treated for M genitalium.Finally, only a patient's first sitafloxacin treatment event during the study period was included in analyses, and subsequent sitafloxacin treatment events were excluded.

Data Analysis
Microbial cure was defined as a negative TOC 14-90 days after completing sitafloxacin.Proportions and 95% confidence intervals (CIs) were calculated by exact methods.Logistic regression models with generalized estimating equations were used to explore patient characteristics associated with treatment failure, accounting for multiple diagnoses from individuals who had M genitalium detected at >1 anatomical site.We assumed an exchangeable correlation structure and used a cluster-based variance estimate for standard error.Known confounders and characteristics with a significance level of P < .05 in univariable analyses were included in the multivariable model, with the following exceptions.Prior treatment failure was reported using 2 different, but correlated measures: (1) number of prior failed antibiotic regimens and (2) prior failure of moxifloxacin.Due to the correlation between the 2 measures, prior treatment failure of moxifloxacin was the variable included in the multivariable model as it had the strongest association with, and greatest clinical relevance to, sitafloxacin failure.We specifically examined correlation and multicollinearity (using variance inflation factors [VIFs]) between independent variables to be included in the multivariable model, and a VIF value ≥2.5 was considered to be an indicator of multicollinearity.When 2 or more variables were deemed to be correlated/colinear, the more clinically relevant variable was included, as described in the Results.We did not include transgender and people who identified as another gender in the logistic regression analyses to avoid bias as there were <5 observations for this variable.
Finally, to evaluate the efficacy of the 2 sitafloxacin regimens used during the study period (ie, sequential monotherapy vs sequential combination therapy), while controlling for prior failure of moxifloxacin, we stratified our study population by prior failure of moxifloxacin versus no prior failure of moxifloxacin.We then assessed the relationship between sitafloxacin regimen and cure within each strata using the Fisher exact test.Stata software (version 17, StataCorp LLC, College Station, Texas) was used for statistical analysis.Ethics approval was obtained from the Alfred Health Ethics Committee (approval number 232/16).

Characteristics of the Study Population
Between 3 January 2017 and 14 February 2022, 306 patients were diagnosed with macrolide-resistant M genitalium at MSHC and treated with a sitafloxacin regimen (Figure 1).Three patients received sitafloxacin monotherapy due to contraindications to doxycycline and were excluded.Of the remaining 303 patients, 250 patients (82.5%) had a TOC recorded within 14-90 days.Twenty-one of 250 cases were excluded: 9 took <50% of the prescribed sitafloxacin doses and 12 cases had a high risk of reinfection.As a result, 229 patients were included in the analysis.A total of 95 of 229 patients received sequential doxycycline followed by sitafloxacin (each drug for 7 days) and 134 received doxycycline followed by sitafloxacin with doxycycline (doxycycline for 14 days, with sitafloxacin for the last 7 days) (Figure 1).
The most common indication for testing was urethritis in men (n = 44 [19.0%]); however, most cases were asymptomatic by the time they received a sitafloxacin regimen (n = 168 [73.4%]).Screening for asymptomatic M genitalium is not practiced or recommended at MSHC.The asymptomatic patients represent those who had initially been symptomatic but had failed other regimens and had persistent asymptomatic infection (n = 156), and a small number (n = 12) who were asymptomatic contacts of patients with resistant infection.

Previous Antibiotic Therapy
Of the 229 patients, 94 (41.1%) had not received prior treatment for their infection (representing patients in the first 5 months of the study period), 70 (30.6%)had failed 1 prior antibiotic regimen, and 65 (28.4%) had failed at least 2 prior regimens.During the first 5 months of the study period, 6% (n = 6/ 93) of patients had failed ≥1 prior regimen.From June 2017, 93% (n = 123/142) had failed ≥1 prior regimen.Of note, 128 (55.9%) patients had previously received and failed 250 patients provided a TOC within 14-90 days Excluded (n = 21): x Took <50% of sitafloxacin doses (n = 9) x High risk of reinfection (n = 12) 229 M genitalium-infected cases treated with sitafloxacin and included in the final analysis 95 patients treated with sequential monotherapy b 134 patients treated with sequential combination therapy c 306 M genitalium cases diagnosed at MSHC and treated with a sitafloxacin regimen between January 2017 and February 2022 Excluded (n = 56): x Lost to follow-up (n = 42) x Returned for TOC outside 14-90 days (n = 6) x Repeat sitafloxacin regimen in the study period (n = 5) x Treated with sitafloxacin alone (n = 3) a Figure 1.Mycoplasma genitalium-infected cases at Melbourne Sexual Health Centre, January 2017-February 2022.a Three patients received sitafloxacin monotherapy 100 mg daily as doxycycline was contraindicated for them.b Sequential monotherapy consisted of doxycycline 100 mg twice daily for 7 days followed by sitafloxacin 100 mg daily for 7 days.c Sequential combination therapy consisted of doxycycline 100 mg twice daily for 2 weeks, with the commencement of sitafloxacin 100 mg daily in the second week of treatment.Abbreviations: MSHC, Melbourne Sexual Health Centre; TOC, test of cure.

Adherence and Adverse Effects
Adherence data were available for 216 (94.3%) cases.Most patients (n = 203 [94.0%]) reported not missing any doses and 13 (6.0%) reported missing between 1 and 6 doses.Tolerability data were available for 211 cases (92.1%); 80 (37.9%) patients reported side effects.Side effects were mild and self-limiting, and no patients ceased treatment early because of side effects.The most common side effects reported were diarrhea (n = 27), nausea (n = 18), and abdominal pain (n = 10).
We included age, gender, prior failure of moxifloxacin, and M genitalium assay in a multivariable model (Table 2).Type of sitafloxacin regimen (ie, sequential monotherapy vs sequential combination therapy) was not included in the model due to high correlation and collinearity with prior failure of moxifloxacin (ρ = −0.844,P < .001),and the latter was selected for inclusion in the model due to its clinical relevance to treatment outcome.As stated, the M genitalium assay used (ie, ResistancePlus MG assay vs Aptima TMA MG assay) was also moderately correlated with prior failure of moxifloxacin (ρ = 0.498, P < .001);however, it was able to be included in  the multivariable model as there was no evidence of multicollinearity (VIF <2.5).Site of infection was not included due to its correlation with gender, and number of prior failed antibiotic regimens was not included due to its correlation with prior failure of moxifloxacin.Overall, in the adjusted analysis, prior failure of moxifloxacin was the only characteristic that remained highly significantly associated with increased odds of sitafloxacin failure (adjusted OR [AOR], 7.56 [95% CI, 2.38-24.04],P < .001).

DISCUSSION
This study reports the efficacy of sitafloxacin for M genitalium infections in the setting of rising macrolide and fluoroquinolone We did not include transgender people and people who identified as another gender in logistic regression analyses to avoid bias as there were <10 observations.c First-pass urine samples were obtained from people who were male at birth.Of the 69 cervicovaginal infections, 53 were detected with cervicovaginal swabs and 16 were detected in first-pass urine samples.resistance and increasingly limited treatment options.The overall cure rate of a sitafloxacin regimen was 80% in our population, with an 8-fold increased odds of failing sitafloxacin in patients who had previously failed moxifloxacin, likely reflecting the presence of existing fluoroquinolone resistance mutations in the parC and/or gyrA genes.Unfortunately, sequencing of these samples was not available due to the impact of coronavirus disease 2019 (COVID-19) on our laboratory services over the study period, but we know that the prevalence of clinically relevant parC mutations exceeded 20% in M genitalium infections in our clinic population during this study [4,10,11].In addition, it is anticipated that approximately 30% of M genitalium infections with a parC mutation will have a concurrent gyrA mutation, further impacting on treatment outcome [7,10].Sitafloxacin cured 94% of M genitalium infections that had not previously failed moxifloxacin compared to 69% of infections that had, highlighting the value of this immediately available information to clinicians to inform of decision making.This could also suggest a lower prevalence of parC and/ or gyrA mutations in the first 5 months of the study, in keeping with data that show this prevalence has slowly increased in our population [10].Our series was unable to establish if sequential combination therapy was superior to sitafloxacin sequential monotherapy due to changes in the indications for sitafloxacin use and a number of highly correlated variables.In particular, prior failure of moxifloxacin was far more common in patients given sequential combination therapy compared to sequential monotherapy.While our stratified analysis was impacted by small numbers, it did suggest that there was likely to be limited or no benefit of sequential combination therapy over sequential monotherapy in patients who had not failed moxifloxacin.In patients who had failed moxifloxacin, it is possible that sequential combination therapy offers some benefit over sequential monotherapy, but larger sample sizes and accompanying parC/gyrA sequencing data are needed to assess this.
Sitafloxacin, a relatively novel fluoroquinolone with bactericidal effects, exhibits in vitro antibacterial activity against gram-positive, gram-negative, and anaerobic bacteria, as well as atypical pathogens [12].It has been shown to have potent activity against multidrug-resistant organisms, displaying lower MICs and lower rates of resistance compared to ciprofloxacin and levofloxacin [13].It has been used to treat respiratory and urinary tract infections in Japan for decades, and became available in Thailand and other parts of the Asia-Pacific in the past decade [14,15].Sitafloxacin is well tolerated, with the most common adverse effect being gastrointestinal disturbance [12].
In adjusted analysis, patients who had previously failed treatment with moxifloxacin were almost 8 times more likely to fail treatment with sitafloxacin compared to those who had not previously failed moxifloxacin, although sitafloxacin still cured 70% of these infections.In the absence of sequencing data, this is likely to be due to specific mutations in the parC gene including the most common parC SNP, G248T, which confers the amino acid change S83I [11,16].In vitro, sitafloxacin exhibits lower MICs in isolates with parC S831 mutations than moxifloxacin [3].Over the past decade at our clinic, there has been a concerning rise in the proportion of M genitalium infections with parC S83I mutations from 13.0% in 2012-2013 to 27.5% in 2019-2020 [10].Importantly, a significant proportion of infections with an S83I mutation also had a concurrent gyrA gene mutation (ie, G285A/M95I), and this has increased from 28.2% in 2016-2018 to 34.8% in 2019-2022 [10].The addition of this gyrA gene mutation to the parC S83I mutation significantly reduces the efficacy of both moxifloxacin and sitafloxacin [10].These data highlight the importance of understanding the prevalence of key fluoroquinolone resistance markers in populations and the increasing value of assays that include relevant SNPs in both genes [4,17] to guide therapeutic decision making for M genitalium, particularly in settings where fluoroquinolone resistance is common.A systematic review and meta-analysis of studies published up to 2019 reported the summary prevalence estimate of mutations associated with fluoroquinolone resistance was 8% worldwide, with the highest prevalence of these mutations in the Western Pacific Region [2].Notably, in Japan there was a significant increase in fluoroquinolone resistance-associated mutations from 5% before 2010 to 29% in 2016-2017 [18], which is similar to what has been observed in our clinic population [10].
Preliminary in vitro data have shown a synergistic effect from the combination of doxycycline and sitafloxacin in M genitalium strains without parC mutations; however, this has not been evaluated for highly resistant strains (J. S. Jensen, personal communication).After our initial small series demonstrating high cure in patients with no further treatment options [5], we hoped to determine if sitafloxacin in combination with doxycycline was superior to sitafloxacin as monotherapy.However, due to the changes in indications for sitafloxacin use and correlation between sequential combination therapy and past failure of moxifloxacin, it was not possible to assess this in regression analyses.While we conducted a stratified analysis, this was significantly impacted by small numbers.Stratified data did suggest that overall cure was high (>90%) in patients who had not previously failed moxifloxacin, regardless of whether sitafloxacin was used as monotherapy or in combination with doxycycline.In patients who had previously failed moxifloxacin, sequential monotherapy cured 33% of infections, while sequential combination therapy cured 71%, suggesting there may be a benefit of sequential combination therapy in heavily pretreated patients.However, as mentioned, small numbers impacted this comparison, and larger sample sizes with sequencing data are needed to determine if sequential combination therapy offers any benefit over sequential monotherapy in heavily pretreated patients with existing resistance.This study has strengths and limitations.MSHC is the largest STI service in Australia, with 60 000 consultations per year, and services a diverse population of >5 million people.All patients who present for a TOC at MSHC see a clinician who uses a template to gather important clinical information, including ongoing symptoms, adherence, and side effects to medications, as well as risk of reinfection.Therefore, these data were collected in a standardized format for all patients.However, these data are self-reported and therefore prone to recall and reporting bias.Additionally, MSHC is a specialist sexual health clinic and findings from this service may not be generalizable to the wider community.Patients who present to sexual health clinics tend to have had higher exposure to prior antibiotics, further increasing the chance of resistance.As mentioned above, during the first 5 months of the study period, sitafloxacin was primarily used firstline for macrolide-resistant M genitalium as sequential monotherapy.In the latter part of the study period, sitafloxacin was used as sequential combination therapy and primarily used for patients who had failed ≥1 prior regimen.As a consequence, a number of variables examined in univariable analyses, including the type of sitafloxacin regimen and failure of prior regimens, were highly correlated and this limited our ability to compare the efficacy of the 2 different sitafloxacin regimens.Furthermore, 2 different M genitalium diagnostic assays were used during the study period, which may have influenced our cure estimates; however, in adjusted analyses, there was no significant difference in the detection of M genitalium between the 2 assays.Finally, the COVID-19 pandemic impacted our service; testing laboratories changed 3 times in this period and we were unable to obtain access to all samples for sequencing of resistance-associated genes.
In conclusion, in a clinic population where the prevalence of clinically relevant parC mutations exceeds 20%, we found that sitafloxacin was well tolerated and cured 81% of macrolide-resistant M genitalium infections, many of which had failed several other regimens.There was a higher likelihood of sitafloxacin failure in patients who previously failed moxifloxacin, but cure still occurred in 70% of these patients, which is important information for clinicians managing patients with limited treatment options.In the context of rising resistance in M genitalium, these data indicate benefit in knowing the prevalence and trends in fluoroquinolone resistance markers in clinic populations and value in incorporating them into assays to help with clinical decision making.

d
Adherence data available for 216 patients (219 infections).e Calculated using 1-sided Fisher exact test.f Prior to March 2021, the ResistancePlus MG assay (SpeeDx) was used for M genitalium testing; after March 2021, transcription-mediated amplification (Aptima MG assay; Hologic Gen-Probe Panther system) was used.

Table 1 . Characteristics of 229 Patients Who Received a Sitafloxacin Regimen Between January 2017 and February 2022
Data are presented as No. (%) unless otherwise indicated.Abbreviations: IQR, interquartile range; MSM, men who have sex with men. a Two had multisite infections (urethral and anorectal).

Table 2 . Characteristics Associated With Failure of Sitafloxacin in Patients With Mycoplasma genitalium Infection a
Abbreviations: CI, confidence interval; IQR, interquartile range; MG, Mycoplasma genitalium; MSM, men who have sex with men; OR, odds ratio.a Two hundred twenty-nine patients infected with MG representing 232 anatomical sites of infection.b

Table 3 . Microbial Cure Following Sequential Monotherapy Versus Sequential Combination Therapy Stratified by Prior Failure of Moxifloxacin
a Calculated using Fisher exact test.

Notes Acknowledgments .
The authors do not have an association that might pose a conflict of interest.Patient consent.This study does not include factors necessitating patient consent.Financial support.C. K. F. and C. S. B. are supported by Australian National Health and Medical Research Council (NHMRC) Leadership Investigator Grants (grant numbers GNT1172900 and GNT1173361, respectively) and E. P. F. C. by an NHMRC Emerging Leadership Investigator Grant (grant number 1172873).
Potential conflicts of interest.All authors: No reported conflicts.