Azithromycin failure in Mycoplasma genitalium urethritis.We report significant failure rates (28%, 95% confidence interval confidence interval, n a statistical device used to determine the range within which an acceptable datum would fall. Confidence intervals are usually expressed in percentages, typically 95% or 99%. 15%-45%) after administering 1 g azithromycin to men with Mycoplasma mycoplasma Any of the bacteria that make up the genus Mycoplasma. They are among the smallest of bacterial organisms. The cell varies from a spherical or pear shape to that of a slender branched filament. genitalium-positive nongonococcal urethritis Nongonococcal Urethritis Definition Any inflammation of the urethra not due to gonorrhea, almost always contracted through sexual intercourse and found far more often in men. . In vitro in vitro /in vi·tro/ (in ve´tro) [L.] within a glass; observable in a test tube; in an artificial environment. in vi·tro adj. In an artificial environment outside a living organism. evidence supported reduced susceptibility of M. genitalium to macrolides. Moxifloxacin administration resulted in rapid symptom resolution and eradication of infection in all cases. These findings have implications for management of urethritis Urethritis Definition Urethritis is an inflammation of the urethra that is usually caused by an infection. Description The urethra is the canal that moves urine from the bladder to the outside of the body. . ********** Mycoplasma genitalium has been well described as a pathogen in men with acute and chronic nongonococcal urethritis (NGU NGU abbr. nongonoccocal urethritis NGU Non-gonococcal urethritis ) and has been associated with cervicitis cervicitis Inflammation of the cervix of the uterus, caused by infection or irritation. It is most common during the years of menstruation. Cervicitis can be acute or chronic and may worsen during pregnancy. It does not cause pain but may lead to polyps. in women (1). Since culturing the organism is difficult, limited information has been available regarding its antimicrobial drug susceptibility. In vitro studies suggest it is susceptible to tetracyclines Tetracyclines Definition Tetracyclines are medicines that kill certain infection-causing microorganisms. Purpose Tetracyclines are called "broad-spectrum" antibiotics, because they can be used to treat a wide variety of , macrolides, and fluoroquinolones (2-4), although reduced susceptibility to tetracyclines (5) and specific fluoroquinolones has been reported (4,6). In clinical studies, doxycycline doxycycline /doxy·cy·cline/ (dok?se-si´klen) a semisynthetic broad-spectrum tetracycline antibiotic, active against a wide range of gram-positive and gram-negative organisms; used also as d. calcium and d. hyclate. and levofloxacin (4,7-11) have substantial failure rates, whereas early reports suggest single-dose azithromycin may be more efficacious (10,11). Treatment guidelines for acute NGU include 1 g single dose of azithromycin or doxycycline for 7 days, but no evidence-based guidelines exist for treatment of M. genitalium-positive NGU. We report treatment failure of single-dose and multidose azithromycin therapy in M. genitalium-positive NGU and provide in vitro evidence of macrolide resistance in clinical isolates. Persistent infection was eradicated with moxifloxacin. The Study Cases were derived from a case-control study case-control study, n an investigation employing an epidemiologic approach in which previously existing incidents of a medical condition are used in lieu of gathering new information from a randomized population. of acute NGU conducted from March 2004 to March 2005 at Melbourne Sexual Health Centre (MSHC MSHC Malchow Schlackman Hoppey & Cooper (Washington, DC) MSHC Mines Safety and Health Commission (United Kingdom) ), Australia (12). Participants completed a questionnaire, underwent examination, and had first-void urine samples analyzed by strand-displacement amplification (ProbeTec-ET CT-Amplified-DNA-Assay, Becton, Dickinson and Company, Sparks, MD, N J, USA) for Chlamydia trachomatis Chlamydia tra·cho·ma·tis n. A species of Chlamydia that causes trachoma, inclusion conjunctivitis, lymphogranuloma venereum, nonspecific urethritis, and proctitis in humans. and by polymerase chain reaction polymerase chain reaction (pŏl`ĭmərās') (PCR), laboratory process in which a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large, readily analyzed sample of a piece of DNA; the process is (PCR PCR polymerase chain reaction. PCR abbr. polymerase chain reaction Polymerase chain reaction (PCR) ) for M. genitalium (13), herpes simplex viruses (HSV-1 and -2), Trichomonas vaginalis Trichomonas vag·i·na·lis n. A protozoan found in the vagina and urethra of women and in the urethra and prostate gland of men. , Ureaplasma urealyticum Ureaplasma urealyticum T strain mycoplasma Microbiology A species of small gram-negative bacteria of the family Mycoplasmataceae that lack a cell wall and catabolize urea–to ammonia; U urealyticum and parvum, Gardnerella vaginalis, and adenoviruses (12). Culture of urethral urethral pertaining to or emanating from urethra. urethral agenesis, urethral atresia failure of development of all or part of the urethra: characterized by complete urine retention. A rare cause of neonatal uremia. samples in modified-Thayer-Martin medium was performed for Neisseria gonorrhoeae. Men with M. genitalium infection were instructed regarding partner notification and reinfection reinfection /re·in·fec·tion/ (-in-fek´shun) a second infection by the same agent or a second infection of an organ with a different agent. re·in·fec·tion n. and were asked to return for a test of cure (TOC) 1 month posttreatment. Men with persistent M. genitalium infection were given 1 g single dose of azithromycin or 1 g weekly for 3 doses, but after apparent failure of azithromycin therapy in 3 men without reinfection, participants with persistent infection were offered moxifloxacin, 400 mg daily for 10 days. Four urethral specimens from men for whom azithromycin therapy failed were inoculated into SP4 medium, frozen (-80[degrees]C), and shipped on dry ice to Statens Serum Institut Statens Serum Institut (English: the State Serum Institute), or SSI for short, is a Danish sector research institute located on the island of Amager in Copenhagen. , Denmark, for culture in Vero cells and antimicrobial drug susceptibility testing (6). M. genitalium strains in Vero cell culture were grown in the presence of different concentrations of antimicrobial drugs, and growth of M. genitalium was monitored by quantitative PCR for determination of MIC (6). The Human Research and Ethics Committee of the Alfred Hospital, Victoria, approved the study. Data were stored in Microsoft Access and analyzed by using SPSS A statistical package from SPSS, Inc., Chicago (www.spss.com) that runs on PCs, most mainframes and minis and is used extensively in marketing research. It provides over 50 statistical processes, including regression analysis, correlation and analysis of variance. version 12 (SPSS Inc., Chicago, IL, USA). Ninety-five percent confidence intervals (CIs) were calculated for proportions, which were compared by using the Fisher exact test. Patients were excluded from the analysis when information or specimens were not available. M. genitalium was detected in 31(9.4%) of 329 patients (95% CI 6.6%--12.9%) and 3 of 307 controls. No patients with M. genitalium infection had other pathogens detected (12). Men with M. genitalium had a median age of 33 years (range 22-54 years); 25 were heterosexual, and 9 were homosexual (behavioral and clinical data are presented elsewhere [12]). Six female and 4 male asymptomatic sexual contacts of M. genitalium-infected men were tested; a throat and anal sample in 1 man and a cervical sample in 1 woman were positive for M. genitalium. Contacts were presumptively treated with 1 g single dose of azithromycin; however, the infected male contact required moxifloxacin after azithromycin treatment failed in this patient and in the index patient. Thirty-two men (94%) completed their TOC, a median of 31 days (range 17-59 days) after receiving azithromycin. Twenty-three (72%) men had a negative TOC (95% CI 55%-85%) and were asymptomatic; however, 9 (28%, 95% CI 15%-45%) were positive for M. genitalium by PCR. No treatment failures reported unprotected sexual contact posttreatment or previous antimicrobial drugs. Azithromycin treatment failed in 4 (44%, 95% CI 16%-76%) homosexual males, compared to 5 (22%, 95% CI 8%-42%) heterosexual males (p = 0.23). Five men for whom azithromycin treatment failed reported sexual contact with partners from Asia before symptom onset (56%, 95% CI 24%-84%) compared to 6 azithromycin responders (27%, 12%-48%), p = 0.22. Eight patients for whom azithromycin treatment failed reported an initial reduction or resolution of symptoms following azithromycin and then experienced recurrent urethral symptoms; 1 male was persistently asymptomatic. The Table outlines urethral Gram stain findings and treatment of men with persistent infection; all 8 men became asymptomatic after receiving moxifloxacin. The 4 TOC specimens from men with azithromycin failure available for culture yielded growth of M. genitalium. Antimicrobial drug susceptibility testing showed increased MICs to macrolides: azithromycin >8 mg/L, erythromyein >32 mg/L, and clarithromycin >32 mg/L. All isolates were susceptible to moxifloxacin (MIC range 0.031-0.125 mg/L) and could be considered susceptible to doxycycline (MIC range 0.125-0.25 mg/L). However, correlates between in vitro MICs and treatment efficacy have not yet been established. Conclusions The azithromycin failure rate in M. genitalium-positive NGU was 28% (15%-45%) in this study and was associated with recurrent urethral symptoms in 8 of 9 cases. Longer course azithromycin ameliorated but did not resolve symptoms or eradicate infection, whereas moxifloxacin resulted in rapid symptom resolution and eradicated infection. Symptom improvement followed by recrudescence recrudescence /re·cru·des·cence/ (re?kroo-des´ens) recurrence of symptoms after temporary abatement.recrudes´cent re·cru·des·cence n. has been reported after levofloxacin failure (9). Culture of M. genitalium from all 4 specimens, and reduced susceptibility to azithromycin in vitro, demonstrates that azithromycin-resistance rather than reinfection caused treatment failure and that nonviable nonviable /non·vi·a·ble/ (-vi´ah-b'l) not capable of living. non·vi·a·ble adj. Not capable of living or developing independently. Used especially of an embryo or fetus. DNA DNA: see nucleic acid. DNA or deoxyribonucleic acid One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. was not the reason for a persistently positive PCR. The availability of strains in pure culture will enable investigation into resistance mechanisms, and work in progress indicates that mutations in region-V of the 23S-rDNA explain the azithromycin resistance (J.S. Jensen, unpub, data). M. genitalium has been associated with persistent NGU (1). Recent data indicate that sequence variation in the gene mediating adhesion to epithelial cells coincides with the immune response in patients and that changes in this gene occur rapidly with persistent infection (14). In vitro studies also suggest that macrolide-resistant mutants can be selected by serial passage of mycoplasmas Mycoplasmas The smallest prokaryotic microorganisms that are able to grow on cell-free artificial media. Their genome size is also among the smallest recorded in prokaryotes, about 5 × 108 to 109 daltons. in subinhibitory concentrations of macrolide (15). Macrolide resistance in our study could have been induced by single-dose azithromycin, which may be suboptimal Suboptimal A solution is called suboptimal if a part of the solution has been optimized without regards to the overall objective. for eradication of a slow-growing bacterium such as M. genitalium. Studies are ongoing to establish whether resistance in our isolates was present pretreatment pretreatment, n the protocols required before beginning therapy, usually of a diagnostic nature; before treatment. pretreatment estimate, n See predetermination. or emerged after azithromycin-exposure. It is possible that initial use of higher doses or longer durations of azithromycin in M. genitalium-positive NGU could avoid selection of resistant mutants. The association between azithromycin failure and sexual partners from Asia may be clinically relevant, given the high levels of antimicrobial drug resistance reported in other sexually transmitted infections such as Neisseria gonorrhoeae infections in Asia, and the higher failure rates seen in homosexual men, while not statistically significant, may represent a core-group effect. Azithromycin or doxycycline is recommended treatment for NGU. While treatment-failure in M. genitalium--positive NGU appears common with doxycycline (4, 7-11), early reports suggest 1 g azithromycin is more effective, with cure rates of 85% (10,11), and that prolonged azithromycin treatment (500 mg on day 1 and 250 mg on days 2-5) eradicates M. genitalium in 95% of cases (10). However, if treatment-failure after 1 g azithromycin is as prevalent as indicated by our study in M. genitalium--positive NGU, this has implications for the use of single-dose azithromycin as first-line treatment for NGU and leaves few evidence-based treatment options. Information regarding sensitivity of M. genitalium to fluoroquinolones has been limited, but reports suggest differential activity against M. genitalium, with levofloxacin (4,9) less active than gatifloxacin, sparfloxacin, and tosufloxacin in vitro and in vivo and moxifloxacin more active than levofloxacin and ciprofloxacin ciprofloxacin /cip·ro·flox·a·cin/ (sip?ro-flok´sah-sin) a synthetic antibacterial effective against many gram-positive and gram-negative bacteria; used as the hydrochloride salt. cip·ro·flox·a·cin n. in vitro (6). We report significant failure rates of azithromycin in M. genitalium-positive NGU that is supported by in vitro evidence of reduced susceptibility to macrolides. Recurrent urethral symptoms following azithromycin therapy only occurred in persons with persistent M. genitalium infection and resolved with moxifloxacin. Because single-dose azithromycin is recommended treatment for NGU, these findings have implications for treatment guidelines and highlight the need for randomized ran·dom·ize tr.v. ran·dom·ized, ran·dom·iz·ing, ran·dom·iz·es To make random in arrangement, especially in order to control the variables in an experiment. studies to determine optimal treatment for M. genitalium-positive NGU and M. genitalium infection in women, who are at high risk for sequelae sequelae Clinical medicine The consequences of a particular condition or therapeutic intervention . Acknowledgments We thank Leonie Horvath, Irene Kuveska, Elice Rudland, and Shujun Chen for laboratory assistance; Mary Santoro for administrative assistance; and Toyota Fonden and Aage Bangs Fond for grants, which partly supported the culturing of M. genitalium culture and determination of antimicrobial drug susceptibility. References (1.) Jensen JS. Mycoplasma genitalium: the aetiological AE`ti`o`log´ic`al a. 1. Pertaining to ætiology; assigning a cause. Adj. 1. aetiological - of or relating to the philosophical study of causation aetiologic, etiologic, etiological 2. agent of urethritis and other sexually transmitted diseases Sexually transmitted diseases Infections that are acquired and transmitted by sexual contact. Although virtually any infection may be transmitted during intimate contact, the term sexually transmitted disease is restricted to conditions that are largely . J Eur Acad Dermatol Venereol. 2004;18:1-11. (2.) Hannan PC, Woodnutt G. In vitro activity of gemifloxacin (SB 265805; LB20304a) against human mycoplasmas. J Antimicrob Chemother. 2000;45:367-9. (3.) Duffy LB, Crabb D, Searcey K, Kempf MC. Comparative potency of gemifloxacin, new quinolones, macrolides, tetracycline tetracycline (tĕ'trəsī`klēn), any of a group of antibiotics produced by bacteria of the genus Streptomyces. They are effective against a wide range of Gram positive and Gram negative bacteria, interfering with protein and clindamycin against Mycoplasma spp. J Antimicrob Chemother. 2000;45(Suppl 1):29-33. (4.) Yasuda M, Maeda S, Deguchi T. In vitro activity of fluoroquinolones against Mycoplasma genitalium and their bacteriological bac·te·ri·ol·o·gy n. The study of bacteria, especially in relation to medicine and agriculture. bac·te efficacy for treatment of M. genitalium-positive nongonococcal urethritis in men. Clin Infect Dis. 2005;41:1357-9. (5.) Hannah PC. Comparative susceptibilities of various AIDS-associated and human urogenital urogenital /uro·gen·i·tal/ (-jen´i-tal) genitourinary. u·ro·gen·i·tal or u·ri·no·gen·i·tal adj. Genitourinary. tract mycoplasmas and strains of Mycoplasma pneumoniae to 10 classes of antimicrobial agent in vitro. J Med Microbiol. 1998;47:1115-22. (6.) Hamasuna R, Osada Y, Jensen JS. Antibiotic susceptibility testing of Mycoplasma genitalium by TaqMan 5' nuclease nuclease /nu·cle·ase/ (noo´kle-as) any of a group of enzymes that split nucleic acids into nucleotides and other products. nu·cle·ase n. real-time PCR. Antimicrob Agents Chemother. 2005;49:4993-8. (7.) Falk L, Fredlund H, Jensen JS. Tetracycline treatment does not eradicate Mycoplasma genitalium. Sex Transm Infect. 2003;79:318-9. (8.) Johannisson G, Enstrom Y, Lowhagen GB, Nagy V, Ryberg K, Seeberg S, et al. Occurrence and treatment of Mycoplasma genitalium in patients visiting STD (Subscriber Trunk Dialing) Long distance dialing outside of the U.S. that does not require operator intervention. STD prefix codes are required and billing is based on call units, which are a fixed amount of money in the currency of that country. clinics in Sweden. Int J STD AIDS. 2000; 11:324-6. (9.) Maeda SI, Tamaki M, Kojima K, Yoshida T, Ishiko H, Yasuda M, et al. Association of Myeoplasma genitalium persistence in the urethra urethra (y  rē`thrə), canal in most mammals that carries urine from the bladder to the outside of the body; in the male it also serves as a genital duct. with recurrence of nongonococcal urethritis. Sex Transm Dis.
2001;28:472-6.(10.) Bjornelius E, Anagrius C, Bojs G, Hans G, Johanisson G, Lidrink P, et al. Mycoplasma genitalium: when to test and treat. Present status in Scandinavia. In: 15th Biennial Meeting of the International Society for Sexually Transmitted Diseases Research. Ottawa, Canada; 2003 [cited 2006 May 22]. Available from http://www.isstdr.org (11.) Mroczowski TF, Mena LA, Nsumai M, Martin DH. A randomized comparison of azithromycin and doxycycline for the treatment of Mycoplasma genitalium-positive urethritis in men. In: 16th Biennial Meeting of the International Society for Sexually Transmitted Diseases Research. Amsterdam, the Netherlands; 2005 [cited 2006 May 22]. Available from http://www.isstdr.org (12.) Bradshaw CS, Tabrizi SN, Read TRH TRH thyrotropin-releasing hormone. TRH abbr. thyrotropin-releasing hormone TRH thyrotropin releasing hormone. , Garland SM, Hopkins CA, Moss LM, et al. Etiologies of non-gonococcal urethritis: bacteria, viruses and the association with oro-genital exposure. J Infect Dis. 2006;193:336-45. (13.) Yoshida T, Deguchi T, Ito M, Maeda S, Tamaki M, Ishiko H. Quantitative detection of Mycoplasma genitalium from first-pass urine of men with urethritis and asymptomatic men by real-time PCR. J Clin Microbiol. 2002;40:1451-5. (14.) Iverson Cabral SL, Astete SG, Cohen cohen or kohen (Hebrew: “priest”) Jewish priest descended from Zadok (a descendant of Aaron), priest at the First Temple of Jerusalem. The biblical priesthood was hereditary and male. CR, Totten PA. Heterogeneity of the MGPB gene in Mycoplasma genitalium, a mechanism for persistence. In: 16th Biennial meeting of the International Society for Sexually Transmitted Diseases Research, Amsterdam, the Netherlands; 2005 [cited 2006 May 22]. Available from http://www.isstdr.org (15.) Pereyre S, Guyot guy·ot n. A flat-topped submarine mountain. [After Arnold Henri Guyot (1807-1884), Swiss-born American geologist and geographer. C, Renaudin H, Charron A, Bebear C, Bebear CM. In vitro selection and characterization of resistance to macrolides and related antibiotics in Mycoplasma pneumoniae. Antimicrob Agents Chemother. 2004;48:460-5. Catriona S. Bradshaw, * ([dagger]) Jorgen S. Jensen, ([double dagger]) Sepehr N. Tabrizi, ([dagger]) ([section]) Timothy R.H. Read, * Suzanne M. Garland, ([dagger]) ([section]) Carol A. Hopkins, * Lorna M. Moss, * and Christopher K. Fairley * ([dagger]) * Alfred Hospital, Victoria, Australia; ([dagger]) University of Melbourne
In 2006, Times Higher Education Supplement ranked the University of Melbourne 22nd in the world. Because of the drop in ranking, University of Melbourne is currently behind four Asian universities - Beijing University, , Victoria, Australia; ([double dagger]) Statens Serum Institut, Copenhagen, Denmark; and ([section]) Royal Women's Hospital, Victoria, Australia Address for correspondence: Catriona S. Bradshaw, Melbourne Sexual Health Centre 580 Swanston St, Carlton, 3053 Victoria, Australia; email: cbradsbaw@msbc.org.au Dr Bradshaw is a physician at Melbourne Sexual Health Centre, Melbourne, Australia. Her current research interests include nongonococcal urethritis, M. genitalium, and bacterial vaginosis.
Table. Case-patients experiencing single-dose azithromycin treatment
failure *
TOC-1
Pretreatment PMN/ Mg
Patient ([dagger]) PMN/HPF HPF PCR
1 ([double dagger]) [greater than or [greater than Pos
equal to] 5 or equal to] 5
2 ([double dagger]) [greater than or < 5 Pos
equal to] 5
3 < 5 [section] Pos
4 ([double dagger]) [greater than or [greater than or Pos
([paragraph]) equal to] 5 equal to] 5
5 ([double dagger]) [greater than or [greater than or Pos
equal to] 5 equal to] 5
6 < 5 [section] Pos
7 (#) [greater than or < 5 Pos
equal to] 5
8 [greater than or < 5 Pos
equal to] 5
9 [greater than or [greater than or Pos
equal to] 5 equal to] 5
TOC-2
PMN/ Mg
Patient ([dagger]) Treatment-2 HPF PCR
1 ([double dagger]) 1 g AZI [greater than or Pos
equal to] 5
2 ([double dagger]) 1 g AZI [section] Pos
3 1 g AZI < 5 Pos
weekly 3
doses
4 ([double dagger]) MOX, 400 mg Neg
([paragraph]) bd 10 d
5 ([double dagger]) MOX, 400 mg Neg
bd 10 d
6 MOX, 400 mg Neg
bd 10 d
7 (#) MOX, 400 mg Neg
bd 10 d
8 MOX, 400 mg Neg
bd 10 d
9 MOX, 400 mg Neg
bd 10 d
TOC-3
PMN/ Mg
Patient ([dagger]) Treatment-3 HPF PCR
1 ([double dagger]) 1 g AZI weekly, < 5 Pos
3 doses
2 ([double dagger]) 1 g AZI weekly, [section] Pos
3 doses
3 MOX, 400 mg [section] Neg
bd 10 d
4 ([double dagger])
([paragraph])
5 ([double dagger])
6
7 (#)
8
9
TOC-4
Mg
Patient ([dagger]) Treatment-4 PCR
1 ([double dagger]) MOX, 400 mg Neg
bd 10 d
2 ([double dagger]) MOX, 400 mg Neg
bd 10 d
3
4 ([double dagger])
([paragraph])
5 ([double dagger])
6
7 (#)
8
9
* PMN/HPF, polymorphonuclear count per high power field (x 1,000
magnification); Mg, Mycoplasma genitalium; PCR, polymerase chain
reaction; Pos, positive; Neg, negative; AZI, azithromycin; MOX,
moxifloxacin; d, days; bd, twice daily; tests of cure (TOCs) were
performed 1 month after commencement of each therapy; TOC-1, first
test of cure 1 month after treatment.
([dagger]) All men treated with 1 g of azithromycin at first
examination.
([double dagger]) Patients with specimens cultured, MIC data available
and presented for all 4 isolates.
([section]) Urethral PMN count not available.
([paragraph]) Patient 4 saw his general practitioner 3 weeks after
receiving 1 g azithromycin with recurrent urethral discharge and
dysuria and was retreated with 1 g azithromycin before his TOC-1.
(#) Only patient who was asymptomatic with persistent infection.
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