Time to Eradication of Mycoplasma genitalium after Antibiotic Treatment in Men and Women
Category: Flocked Swabs
Publication Date: 08/17/2015
Conference or Journal: Journal of Antimicrobial Chemotherapy
Author(s): Department of Dermatology and Venereology, RegionO¨ stergo¨ tland, Linko¨ping, Sweden; Department of Clinical and Experimental Medicine, Linko¨ping University, SE-581 85 Linko¨ping, Sweden; R&D Department of Local Health Care, Region O¨ stergo¨ tland, SE-581 85 Linko¨ping, Sweden; Department of Dermatology and Venereology, Va¨stervik Hospital, SE-593 81 Va¨stervik, Sweden; Microbiology and Infection Control, Sexually Transmitted Infections, Research and Development, Statens Serum Institut, Copenhagen, DK-2300 København S, Denmark
Objectives: The objectives of this study were to evaluate the time to a Mycoplasma genitalium-negative test after start of treatment and to monitor if and when antibiotic resistance developed.
Methods: Sexually transmitted disease (STD) clinic attendees with suspected or verified M. genitalium infection were treated with azithromycin (5 days, 1.5 g; n¼85) or moxifloxacin (n¼5). Subjects with symptomatic urethritis or cervicitis of unknown aetiology were randomized to either doxycycline (n¼49) or 1 g of azithromycin as a single dose (n¼51). Women collected vaginal specimens and men collected first-catch urine 12 times during 4 weeks. Specimens were tested for M. genitalium with a quantitative MgPa PCR and for macrolide resistance-mediating mutations with a PCR targeting 23S rRNA. Clinical Trials Registration: NCT01661985.
Results: Ninety M. genitalium cases were enrolled. Of 56 patients with macrolide-susceptible strains before treatment with azithromycin (1.5 g, n¼46; 1 g single oral dose, n¼10), 54 (96%) had a negative PCR test within 8 days. In four patients, M. genitalium converted from macrolide susceptible to resistant after a 10 day lag time with negative tests (azithromycin 1.5 g, n¼3; 1 g single oral dose, n¼1). Moxifloxacin-treated subjects (n¼4) were PCR negative within 1 week. Six of eight (75%) remained positive despite doxycycline treatment.
Conclusions: PCR for M. genitalium rapidly became negative after azithromycin treatment. Macrolide-resistant strains were detected after initially negative tests. Test of cure should be recommended no earlier than 3–4 weeks.
Mycoplasma genitalium is a common pathogen causing sexually transmitted infections (STIs). In contrast to Chlamydia trachomatis, antibiotic resistance is common. Despite reports of excellent eradication rates by azithromycin treatment (85%–95%) in studies recruiting patients before 2005,1–3 there is now growing evidence of decreased susceptibility to macrolides.4–8 Macrolide resistance following a single 1 g dose of azithromycin has been frequently reported and has been suspected to be the major cause of resistance in the community.4,9–11 The resistance is caused by single base mutations in region V of the 23S rRNA gene.12 In most STI treatment guidelines, doxycycline or 1 g of azithromycin as a single oral dose is the recommended treatment for chlamydial infections as well as for non-gonococcal urethritis and cervicitis of unknown aetiology.13,14 First-line treatment of M. genitaliumin Scandinavia is an extended azithromycin course (500 mgon the first day followed by 250 mg on days 2–5). If macrolide resistance occurs, the currently accepted effective option is moxifloxacin treatment with 400 mg once daily for 7–10 days. Due to emerging resistance, a test of cure is recommended. There is, however, no available information regarding how long it takes until M. genitalium is cleared after antibiotic treatment has started. The aims of this prospective trial were to evaluate the time after initiation of treatment to a negative test, and to monitor if and when antibiotic resistance develops.
The study was a prospective longitudinal cohort study comprising an observational study and a randomized treatment trial. From a research point of view, a randomized trial, preferably comprising four arms [doxycycline, 1 g of azithromycin, extended azithromycin (1.5 g) and moxifloxacin], would have been ideal. However, randomizing patientswith a high suspicion of M. genitalium infection would not have been ethically sound. Doxycycline eradicates M. genitalium from only 30% of patients1 –3 and
use ofmoxifloxacin should be limited to infections where other alternatives are unavailable due to the risk of serious adverse events. Consequently, patients were enrolled in two groups in the study: Group 1 comprised patients with a high risk of M. genitalium infection, i.e. with a current partner having an M. genitalium infection or patients not treated initially, but with a positive M. genitalium test. These patients were treated with extended azithromycin. If there was evidence of macrolide resistance, moxifloxacin was prescribed. Group 2 comprised symptomatic patients with no known exposure to
M. genitalium. These patients were randomized to receive doxycycline (200 mg on day 1 and 100 mg once daily on days 2–9; total of 1 g) or to a 1 g single dose of azithromycin, on the day of the first visit.
A substantial proportion of these patients had a chlamydial infection or idiopathic urethritis and/or cervicitis. These patients will not be included in the present analysis. Patients were randomized to either of the two treatment arms by opening the next consecutively numbered envelope containing information regarding treatment.
Subjects and specimens
All patients attending two sexually transmitted disease (STD) clinics from 2010 to February 2014 with a verified or a suspected M. genitalium infection were eligible for enrolment. Exclusion criteriawere allergy to antibiotics used in the study and inability to understand the study procedure. Informed consent was obtained by a nurse or a physician, who also gave the patient oral and written information. The regional research ethics committee of Linko¨ping, Sweden, approved the study on 12 August 2009 (M 134-09, T 126-09). According to the Biobanks in Medicine Care Act (2002:297) a biobank was registered in the Department of Dermatology and Venereology, RegionO¨ stergo¨ tland. Clinical Trials Registration: NCT01661985. In the clinic, a genital smear was examined by microscopy. Samples
were first-catch urine from men and endocervical/vaginal swabs, which were collected in GeneLock transport medium (Sierra Diagnostics, Sonora, CA, USA) for analysis of M. genitalium and C. trachomatis. All laboratory analyses were performed at Statens Serum Institut (SSI) in Copenhagen. The participating men were given 12 tubes with GeneLock urine transport medium, plastic cups, labels, written information and four prestamped envelopes for weekly mail transport to SSI. They were asked not to micturate within 1 h before sampling. Similarly, female participants were provided with 12 Copan flocked swabs (Copan, Brescia, Italy) and tubes with GeneLock swab transport medium.
All participants were asked to collect samples 1, 3, 5, 8, 10, 12, 15, 17, 19, 22, 24 and 26 days after the first day of antibiotic treatment. They were informed not to have any unprotected sexual intercourse and to inform their current sexual partners to attend an STD clinic for testing and antibiotic
M. genitalium was detected by a quantitative PCR amplifying a part of the MgPa gene as previously described;15 in brief, 1.8 mL of the urine sample was concentrated by centrifugation and DNA was released from the pellet by boiling in 300 mL of a 20% (w/v) Chelex 100 slurry (Bio-Rad, Richmond,
CA, USA) in TE buffer [10mM Tris–HCl (pH 8.0)/1mM EDTA]. Similarly, 100 mL of the vaginal swab sample was added to 300 mL of Chelex slurry. All positive results were confirmed in a conventional PCR amplifying the 23S rRNA gene and amplicons were subjected to sequencing on a PyroMark Q96 (Qiagen, Hilden, Germany) sequencing platform.8 Strain typing from selected M. genitalium-positive samples was carried out using an MgPa typing assay.16 Statistical analyses All data were registered in an Excel book using a coded identity for all participants. Kaplan–Meier survival plots were generated and log-rank analysis was performed in GraphPad Prism v6.03.
Of the 191 enrolled subjects, 100were allocated to randomization (Group 2) and 22 (22%) of these were M. genitalium positive. The remaining 91 [68 (75%) M. genitalium positive] were allocated to either the extended azithromycin or the moxifloxacin treatment arm (Group 1). A flow chart of allocation and treatment is presented in Figure 1. Twenty-five men and 37 women were treated with extended azithromycin, 3 men and 2 women were treated with moxifloxacin, 10 men and 5 women were allocated to 1 g of azithromycin treatment as a single dose, and 6 men and 2 women were treated with doxycycline. Demographic data are presented in Table 1. All 12 follow-up specimens were collected and
analysed from 37 (80%) women and 16 (36%) men. More than six specimens were submitted by a further five women (11%) and 14 men (32%). Among the 28 men harbouring a macrolidesusceptible M. genitalium strain, 13 submitted all samples and 7 submitted at least eight samples (mean 9.3), with the last sample taken between days 17 and 26. Observational study of M. genitalium-infected subjects Fifty-three M. genitalium-infected patients, 18men and 35 women, treated with extended azithromycin (1.5 g), were evaluable, and, of these, 33 (62%) had negative tests by day 3 after starting treatment
(pattern A, Figure S1, available as Supplementary data at JAC Online) and remained negative in the remaining tests during the study period of 26 days. Of 10 patients with macroli desusceptible M. genitalium infections treated with 1 g of azithromycin as a single dose, 9 cleared the infection before day 8. There was no gender difference in the clearance rate. The four patients treated with moxifloxacin cleared their infections before day 3, whereas among the seven evaluable subjects treated with doxycycline, three became M. genitalium negative within 1 week. In one of these (a man) symptoms recurred and the tests became M. genitalium positive on day 13. Comparison of outcomes between doxycycline and azithromycin For subjects without pre-existing macrolide resistance there was no difference in time to eradication between 1 g of azithromycin and extended azithromycin (1.5 g) treatment, whereas the difference between azithromycin and doxycycline was statistically significant
(P¼0.03 compared with 1 g and P¼0.006 compared with extended azithromycin) (Figure 2). When all subjects regardless of pre-existing macrolide resistance were compared, only extended azithromycin had a shorter time to eradication than doxycycline (P¼0.04).
Figure 1. Flow chart of all enrolled M. genitalium-positive participants (n¼90), allocation and outcome of antibiotic treatment. Dropouts (n¼13) are defined as those participants who did not collect any sample or did not send them to SSI for analysis. aA man enrolled because of his current partner’s
M. genitalium infection. The physician prescribed 1 g of azithromycin as a single oral dose instead of a 5 day azithromycin treatment by mistake. bA woman stopped the treatment due to nausea after 1 day. She was then treated with extended azithromycin and it turned out that the M. genitalium strain was susceptible to macrolides. The patient had negative tests the day after the azithromycin treatment commenced and throughout the 26 day period. cThree subjects had a macrolide-resistant M. genitalium strain initially and three developed macrolide resistance during or after azithromycin treatment. dThree subjects had a macrolide-resistant M. genitalium strain initially and one developed macrolide resistance during or after azithromycin treatment. eOne man had symptoms of dysuria and scrotal discomfort and recurrent M. genitalium-positive specimens (after a week of negative tests). 1 g AZM, 1 g of azithromycin as a single dose; AZM 5 days, extended azithromycin for 5 days (500+250 mg×1 for 4 days); MXF, moxifloxacin (400 mg×1) for 7 days; DOX, 200 mg of doxycycline on day 1 followed by 100 mg daily for 8 days; Mg pos, M. genitalium positive; Mg neg, M. genitalium negative.
Possible spontaneous clearance of M. genitalium infections
Most of the non-randomized subjects were enrolled due to a positive test. Thus, there was a delay between the initial visit to the clinic and the first day of treatment. Six women and three men were M. genitalium negative in the enrolment test and the following samples. Three subjects (33%) had macrolide-resistant M. genitalium strains in specimens collected before enrolment. Emergence of macrolide resistance following treatment with azithromycin Two women treated with extended azithromycin and one woman treatedwith a single dose of 1 g of azithromycin and with macrolide susceptible strains cleared their infections within 5 days, but after 2 weeks sporadic specimens that were positive, with a few DNA copies of a macrolide-resistant strain (A2058G mutation), began to
appear until M. genitaliumwas persistently detectedwith an increasing organism load with the A2058G mutation in consecutive samples (Table 2) (pattern D, Figure S1). One symptomatic man with a high M. genitalium load at inclusion converted from susceptible to macrolide resistant (A2058G) on day 5 (pattern B, Figure S1). All four subjects denied sexual intercourse during the study period and all MgPa strain types for each subject were identical. There was no difference in the M. genitalium load in the pre-treatment samples between patients developing resistance and those who cleared the infection, with a mean pre-treatment bacterial load of 9786 copies (per 5 mL of extracted DNA) and 7355 copies, respectively.
The present prospective observational study showed that M. genitalium was eradicated within a few days after antibiotic treatment started. For some patients, however, even with extended azithromycin treatment conversion from macrolide-susceptible to macrolide-resistant strains occurred and in most cases it occurred after consecutive negative samples. Among the few patients treated with doxycycline, the clearance rate was low and in line with other studies.1–3,10 Previous studies have reported a slow clearance of C. trachomatis with persistent positive nucleic acid amplification tests for 3 weeks.17,18 M. genitalium DNA would be expected, consequently, to be detectable for weeks, but this was clearly not
the case. One explanation could be that the organism load among M. genitalium-infected patients is 100-fold lower than in C. trachomatis-infected patients.19 Despite the low M. genitalium DNA load, however, the proportion of symptomatic patients was similar to that reported for patients with chlamydia (Table 1).20,21 One man treated with doxycycline had a temporary clearance of M. genitalium DNA, but a subsequent relapse. This was probably due to a temporary suppression of the infection with DNA shedding below the limit of detection. This is in good agreement with previous observations of a temporary effect of tetracyclines and subsequent relapse.2 There is little evidence fromin vitro studies
that tetracycline resistance is common despite frequent treatment failure.22 In four azithromycin-treated subjects, macrolide resistance developed during or after treatment. Obviously, it is impossible
to exclude the possibility of re-infection from an untreated partner, but DNA typing clearly suggested that it was the same strain, and the participants denied any risk of a new infection during the study period. In contrast to other studies, we did not find a higher M. genitalium organism load in patients developing resistance compared with those who cleared the infection, but the number of patients developing resistance was small.23,24 Many studies have shown an overall good response to azithromycin treatment, but there have been reports of resistance emerging after single dose azithromycin treatment.8,10,11 Development of resistance in 3 (5%) of 53 patients treated with extended 1.5 g azithromycin is in good agreement with previous experience of treatment failure
in 5% of patients treated with this dosage.1 However, it also stresses that the extended dosage does not preclude resistance development. With only a few patients treated with a single dose of 1 g of azithromycin, the figures are too small to allow an estimation of differences in the risk for development of resistance between the two regimens. A study by Anagrius et al.,10 however, suggested that single-dose azithromycin was more likely to select for macrolide resistance than the extended dosage
scheme and no development of resistance among 77 patients receiving extended azithromycin was observed. However, 52 of these patients had been treated with doxycycline prior to the azithromycin course, which may partly explain this discrepancy in the risk of macrolide resistance development in two populations that would be considered very similar. Emerging macrolide resistance has been reported in Sweden, from 0% in 2006 to 20% in 2011.10 In Sweden, doxycycline is recommended as first-line treatment for chlamydia and the adherence to this recommendation is very high. Azithromycin is in general only prescribed when there is a verified or high suspicion of M. genitalium infection. The easy access to diagnosis of M. genitalium infection in Sweden and the increasing awareness of the infection means that previously undetected asymptomatic cases now are treated. This might increase the risk of development of antibiotic resistance, especially since most laboratories do not test for macrolide resistance-mediating mutations. Consequently, resistant strains may have a higher risk of being transmitted in the community. The use of moxifloxacin for patients failing macrolide treatment
has been described as very efficient in the initial publications.4,25 Due to uncommon but severe adverse events, moxifloxacin treatment may be ethically problematic for first-line treatment, especially
in cases of asymptomatic infections. In a recently published Australian study there was evidence of emerging antibiotic resistance to moxifloxacin as well, dramatically reducing the options for treatment.11 Spontaneous eradication probably occurred in several patients in the present study. Whether such clearance improves the immune response and diminishes the risk of re-infection, as has
been suggested in women treated for chlamydia, is unclear and not studied.26 Apart from treatment of symptoms, the reason for treatment is to avoid sexual transmission and prevent salpingitis. Most treatment regimens in pelvic inflammatory disease treatment guidelines do not cover M. genitalium, and this may increase the risk of tubal factor infertility or extra-uterine pregnancies. 14 Although spontaneous eradication may occur, studies have suggested that M. genitalium may persist for more than a year.7,27 Consequently, efficient treatment is essential to prevent spread of the infection.
Among the subjects in the present study, there was a very low proportion (11%) not collecting samples. Although the subjects were informed about the importance of not having any unprotected sex during the sampling period, it cannot be ruled out that the adherence was ,100%. We did not have any control of adherence to antibiotic intake (except for those prescribed 1 g of azithromycin as a single dose) and there may be participants not completing the treatment. A few individuals had a few sporadic positive tests, but in these cases the remaining tests were negative, suggesting spontaneous eradication.
In conclusion, the present study indicated that the commonly used recommendation of test of cure 3–4 weeks after the start of treatment can still be used. This is in accordance with a Japanese study where the optimal time for test of cure was found to be 20 days after the start of treatment.28 The present study raises concern that first-line treatment with extended azithromycin for 5 days (1.5 g), as commonly recommended, may not be optimal. With the increasing level of pre-existing macrolide resistance in most settings, treatment failure will be common. Moxifloxacin treatment was effective and may still be an alternative for second-line treatment of macrolide-resistant infections. However, the emergence of moxifloxacin-resistant strains may also threaten the use of this drug. In the absence of routine testing for macrolide resistance-mediating mutations, a test of cure 3–4 weeks after treatment is highly recommended.
We are most grateful to the staff at the STD clinics in Va¨ stervik and Norrko¨ ping (especially to Kajsa Lindstro¨m, Jessica So¨derlind, Margreth Wastesson, Marie Karlsson, Jeanette Groenheit and Zarah Ragnsa¨ ter) and also to Birthe Dohn, Susanne Cramer Johansson and Elvira Chapka for excellent technical assistance in analysing the samples at SSI.
This work was partly supported by the Medical Research Council of
Southeast Sweden, ALF grants from the County Council of O¨ stergo¨ tland
and by Linko¨ pings La¨karesa¨ llskap (Linko¨ ping Society of Medicine),
None to declare.
L. F. initiated the study, examined and sampled most of the patients
in Norrko¨ ping, collected all data and wrote the first draft of the
manuscript. M. E. examined patients in Va¨ stervik and contributed to
amendments to the manuscript. J. S. J. was responsible for the laboratory
tests performed at SSI in Copenhagen and contributed to the design of the
study, analysis of the data and amendments to the manuscript.
Figure S1 is available as Supplementary data at JAC Online (http://jac.
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