New Detection of Locally Acquired Japanese Encephalitis Virus Using Clinical Metagenomics, New South Wales, Australia.

In the context of an emerging Japanese encephalitis outbreak within Australia, we describe a novel locally acquired case in New South Wales. A man in his 70s had rapidly progressive, fatal meningoencephalitis, diagnosed as caused by Japanese encephalitis virus by RNA-based metagenomic next-generation sequencing performed on postmortem brain tissue.

Emerging Genotype IV Japanese Encephalitis Virus Outbreak in New South Wales, Australia.

The detection of a new and unexpected Japanese encephalitis virus (JEV) outbreak in March 2022 in Australia, where JEV is not endemic, demanded the rapid development of a robust diagnostic framework to facilitate the testing of suspected patients across the state of New South Wales (NSW). This nascent but comprehensive JEV diagnostic service encompassed serological, molecular and metagenomics testing within a centralised reference laboratory. Over the first three months of the outbreak (4 March 2022 to 31 May 2022), 1,061 prospective samples were received from 878 NSW residents for JEV testing. Twelve confirmed cases of Japanese encephalitis (JE) were identified, including ten cases diagnosed by serology alone, one case by metagenomic next generation sequencing and real-time polymerase chain reaction (RT-PCR) of brain tissue and serology, and one case by RT-PCR of cerebrospinal fluid, providing an incidence of JE over this period of 0.15/100,000 persons in NSW. As encephalitis manifests in <1% of cases of JEV infection, the population-wide prevalence of JEV infection is likely to be substantially higher. Close collaboration with referring laboratories and clinicians was pivotal to establishing successful JEV case ascertainment for this new outbreak. Sustained and coordinated animal, human and environmental surveillance within a OneHealth framework is critical to monitor the evolution of the current outbreak, understand its origins and optimise preparedness for future JEV and arbovirus outbreaks.

Emergence of Japanese encephalitis in Australia: a diagnostic perspective.

The unprecedented emergence of Japanese encephalitis (JE) in mainland Australia represents an outbreak of high clinical and public health significance. JE is a zoonosis spread by mosquitoes and is one of the most important causes of endemic viral encephalitis in South-East Asia and the Indian subcontinent. While occasional cases of human Japanese encephalitis virus (JEV) infection have occurred in far north Australia, its detection in pigs and the substantial number of locally acquired human cases across multiple jurisdictions in early 2022 prompted the declaration of this outbreak as a Communicable Disease Incident of National Significance. Laboratory testing for JEV is complex, and most cases are diagnosed by serology, for which interpretation is difficult. This review provides a comprehensive outline of currently available methods for JEV diagnosis including serology, nucleic acid amplification testing, virus isolation, sequencing and metagenomics. The relative advantages and disadvantages of the diagnostic tests are presented, as well as their value in clinical and public health contexts. This review also explores the role of mosquito, veterinary and human surveillance as part of the laboratory response to JEV. As JEV may become endemic in Australia, a collaborative and coordinated One Health approach involving animal, human and environmental health is required for optimal disease response and control.

Mycoplasma genitalium coinfection in men with symptomatic gonococcal urethritis.

OBJECTIVES: International guidelines recommend Mycoplasma genitalium testing, preferably using an assay to detect macrolide resistance-associated mutations, for men presenting with non-gonococcal urethritis, but there is no specific guidance on such testing for men with gonococcal urethritis. METHODS: This study aimed to estimate the proportion of men with gonococcal urethritis who have coinfection with M. genitalium through a retrospective analysis of cases of symptomatic urethral gonorrhoea at Western Sydney Sexual Health Centre in 2017 and 2018. RESULTS: Fourteen of 184 (7.6%, 95% CI 3.7 to 11.5) men with gonococcal urethritis had M. genitalium detected in the urine at the time of presentation. No demographic or behavioural factors predicted M. genitalium coinfection. Coinfection with urethral Chlamydia trachomatis was detected in 29 of 184 (15.8%, 95% CI 10.5 to 21.1). All five men with macrolide-resistant M. genitalium detected returned for treatment with moxifloxacin at a median of 8 days (range 5-16 days) after presentation and treatment of gonorrhoea; three of five were documented to remain symptomatic at this visit. CONCLUSION: Although M. genitalium coinfection is less common than chlamydia among men with symptomatic gonococcal urethritis, M. genitalium testing, using an assay to detect macrolide resistance, will potentially reduce symptom duration particularly for men with macrolide-resistant infections, but may not be justifiable on cost-benefit analysis.

Meningococcal and pneumococcal carriage in Hajj pilgrims: findings of a randomized controlled trial.

BACKGROUND: Intense congestion during the Hajj pilgrimage amplifies the risk of meningococcal carriage and disease, and there have been many meningococcal outbreaks reported amongst pilgrims. Thus, a strict vaccination policy is enforced by the host country and either polysaccharide or conjugate quadrivalent meningococcal vaccines are mandatory. However, unlike conjugate vaccines, the polysaccharide vaccine is not thought to reduce pharyngeal carriage of meningococci. METHODS: A single-blinded, randomized, controlled trial amongst pilgrims from Saudi Arabia and Australia during the Hajj seasons of 2016-2017 was conducted to compare MenACWY-Conjugate vaccine with MenACWY-Polysaccharide vaccine, to determine if the conjugate vaccine is more effective in reducing asymptomatic carriage of meningococci, and whether the effect may be long-standing. Oropharyngeal swabs were obtained pre-, immediately post- and 6-11 months following completion of Hajj and tested for the presence of meningococci. RESULTS: Amongst 2000 individuals approached, only 1146 participants aged 18-91 (mean 37.6) years agreed to participate and were randomized to receive either the polysaccharide (n = 561) or the conjugate (n = 561) vaccine, 60.8% were male, and 93.5% were from Saudi Arabia. Amongst oropharyngeal swabs obtained before Hajj, only two (0.2%) tested positive for Neisseria meningitidis. Similarly, meningococci were identified in only one sample at each of the post-Hajj and late follow-up visits. None of the carriage isolates were amongst the serogroups covered by the vaccines. A post hoc analysis of the third swabs revealed that 22.4% of all participants (50/223) were positive for Streptococcus pneumoniae nucleic acid. CONCLUSION: The low overall carriage rate of meningococci found amongst Hajj pilgrims in 2016 and 2017 demonstrates a successful vaccination policy, but neither supports nor refutes the superiority of meningococcal conjugate ACWY vaccine over the polysaccharide vaccine against carriage. Although an association could not be established in this study, molecular epidemiology would help to establish the role of Hajj in facilitating transmission of pneumococci and inform vaccination policy.

Deep Sequencing of Urine Specimens Detects Two BK Polyomavirus Genotypes in a Hematopoietic Stem Cell Transplant Recipient.

BK polyomavirus (BKPyV) is an important pathogen in transplant recipients. We report four draft BKPyV genomes, three of BKPyV genotype I (subtype I-b2) (AUS-105, AUS-106, and AUS-108) and one of genotype II (AUS-107). These draft genomes were identified in longitudinal urine samples collected from a single hematopoietic stem cell transplant recipient.

Prevalence of rectal Mycoplasma genitalium and macrolide resistance in men who have sex with men attending Sydney Sexual Health Centre.

Background Sexually transmissible infections (STIs) have been increasing in men who have sex with men (MSM) in recent years; however, few studies have investigated the prevalence or antimicrobial resistance in rectal Mycoplasma genitalium in this group. This study aimed to determine the prevalence and predictors of rectal M. genitalium in MSM attending an urban sexual health service in Sydney, Australia, namely the Sydney Sexual Health Centre (SSHC), as well as estimate the rate of macrolide resistance. METHODS: A prospective cross-sectional analysis was conducted of rectally asymptomatic MSM having a rectal swab collected as part of their routine care. Participants self-collected a rectal swab to be tested for M. genitalium and completed a 14-item questionnaire that provided information on behavioural risk factors. The prevalence of rectal M. genitalium was determined and multivariate analysis was performed to assess the associations for this infection. Positive specimens then underwent testing for macrolide-resistant mutations (MRMs) using the ResistancePlus MG assay (SpeeDx, Eveleigh, NSW, Australia). RESULTS: In all, 742 patients were consecutively enrolled in the study. The median age was 31 years (interquartile range 27-39 years), with 43.0% born in Australia. Overall, 19.0% of men were bisexual, 22.9% were taking pre-exposure prophylaxis (PrEP) and 4.3% were HIV positive. The prevalence of rectal M. genitalium was 7.0% (95% confidence interval (CI) 5.3-9.1) overall and 11.8% in those taking PrEP. On multivariate analysis, PrEP use was significantly associated with having rectal M. genitalium (odds ratio 2.01; 95% CI 1.09-3.73; P = 0.01). MRMs were detected in 75.0% (36/48; 95% CI 60.4-86.4%) of infections. CONCLUSION: Rates of rectal M. genitalium infection were high among asymptomatic MSM attending SSHC and MRMs were detected in 75% of infections. PrEP use was found to be significantly associated with rectal M. genitalium infection. These data contribute to the evidence base for screening guidelines in MSM.

Men Who Have Sex With Men With Mycoplasma genitalium-Positive Nongonococcal Urethritis Are More Likely to Have Macrolide-Resistant Strains Than Men With Only Female Partners: A Prospective Study.

BACKGROUND: Mycoplasma genitalium was previously less common among men who have sex with men (MSM) compared with men with only female partners (MSW) in men with nongonococcal urethritis (NGU) in Sydney, Australia. We aimed to determine the prevalence of M. genitalium and of macrolide-resistant M. genitalium in men with NGU and to compare differences between prevalence and resistance rates between MSM and MSW. METHODS: We enrolled 588 men with NGU in a prospective study at two urban sexual health services. The ResistancePlus MG assay (SpeeDx, Australia) was used to detect both M. genitalium, and macrolide resistance-associated mutations in first-void urine samples. Demographic, behavioral and clinical data were analyzed to investigate associations with M. genitalium infection or the presence of macrolide resistance. RESULTS: Mycoplasma genitalium prevalence was 12.8% (75 of 588) overall and among MSM (12.8% [39 of 306]) and MSW (12.8% [36 of 282]; risk ratio [RR], 1.00; 95% confidence interval [CI], 0.65-1.52). Overall, 70.7% (53 of 75) of M. genitalium strains were macrolide-resistant, with significantly more resistance among MSM (89.7%, 35 of 39) than MSW (50%, 18 of 36) (RR, 1.80; 95% CI, 1.27-2.54; P = 0.001). On multivariate analysis, the presence of M. genitalium macrolide resistance mutations was independently associated with having male sexual partners compared with having only female partners (RR, 1.55; 95% CI, 1.02-2.38; P = 0.042). CONCLUSIONS: Prevalence of M. genitalium among men with NGU is now similar for MSW and MSM and has increased locally from 5.2% to 12.8% within the last 10 years. Men who have sex with men are significantly more likely than MSW to harbor macrolide-resistant M. genitalium infections. This has treatment implications.

Mycoplasma genitalium: high prevalence of resistance to macrolides and frequent anorectal infection in men who have sex with men in western Sydney.

OBJECTIVES: We aimed to estimate the prevalence of Mycoplasma genitalium infection and of mutations linked to macrolide resistance using the ResistancePlus MG assay (SpeeDx, Sydney, New South Wales, Australia) in first-void urine (FVU), anorectal and oropharyngeal samples from men who have sex with men (MSM) attending Western Sydney Sexual Health Centre (WSSHC). METHODS: Consecutive symptomatic and asymptomatic MSM attending for STI testing were prospectively enrolled. M. genitalium testing using the ResistancePlus MG assay was performed on FVU, anorectal and oropharyngeal samples routinely collected for Chlamydia trachomatis and Neisseria gonorrhoeae assays. RESULTS: Overall, the prevalence of M. genitalium infection in the study group was 13.4% (68/508). Most (79.4%, 54/68) M. genitalium harboured macrolide resistance mutations (87.5% of urethral and 75.6% of anorectal infections). The anorectum was the most commonly infected site (45/505, 8.9%), followed by the urethra (24/508, 4.7%). No oropharyngeal M. genitalium infections were detected (0/508). Most of the anorectal (93.3%) and urethral (79.2%) infections were asymptomatic.MSM who were taking HIV pre-exposure prophylaxis (PrEP) were twice as likely to be infected with M. genitalium compared with MSM who were not on PrEP (OR 2.1, 95% CI 1.3 to 3.6; P=0.0041). Always using condoms for anal sex in the last 3 months was protective of infection (OR 0.8, 95% CI 0.6 to 1.0; P=0.0186). CONCLUSIONS: We demonstrated a high prevalence of M. genitalium and very high levels of macrolide resistance among MSM attending WSSHC. Our findings support the routine use of an assay to detect macrolide resistance mutations in M. genitalium infections. This will ensure, in regions or populations with high rates of macrolide resistance among M. genitalium strains, that first-line treatment with azithromycin will only be used if a macrolide-sensitive strain is identified.

Genome Sequencing Links Persistent Outbreak of Legionellosis in Sydney (New South Wales, Australia) to an Emerging Clone of Legionella pneumophila Sequence Type 211.

The city of Sydney, Australia, experienced a persistent outbreak of Legionella pneumophila serogroup 1 (Lp1) pneumonia in 2016. To elucidate the source and guide public health actions, the genomes of clinical and environmental Lp1 isolates recovered over 7 weeks were examined. A total of 48 isolates from human cases and cooling towers were sequenced and compared using single-nucleotide polymorphism (SNP)-based core-genome multilocus sequencing typing (MLST) and pangenome approaches. All three methods confirmed phylogenetic relatedness between isolates associated with outbreaks in the Central Business District (CBD) in March and May and those in suburb 1. These isolates were designated the "main cluster" and consisted of isolates from two patients from the CBD March outbreak, one patient and one tower isolate from suburb 1, and isolates from two cooling towers and three patients from the CBD May outbreak. All main cluster isolates were sequence type 211 (ST211), which previously has only been reported in Canada. Significantly, pangenome analysis identified mobile genetic elements containing a unique type IV A F-type secretion system (T4ASS), which was specific to the main cluster, and cocirculating clinical strains, suggesting a potential mechanism for increased fitness and persistence of the outbreak clone. Genome sequencing enabled linking of the geographically dispersed environmental sources of infection among the spatially and temporally coinciding cases of legionellosis in a highly populated urban setting. The discovery of a unique T4ASS emphasizes the role of genome recombination in the emergence of successful Lp1 clones.IMPORTANCE A new emerging clone has been responsible for a prolonged legionellosis outbreak in Sydney, Australia. The use of whole-genome sequencing linked two outbreaks thought to be unrelated and confirmed the outliers. These findings led to the resampling and subsequent identification of the source, guiding public health actions and bringing the outbreak to a close. Significantly, the outbreak clone was identified as sequence type 211 (ST211). Our study reports this ST in the Southern Hemisphere and presents a description of ST211 genomes from both clinical and environmental isolates. A unique mobile genetic element containing a type IV secretion system was identified in Lp1 ST211 isolates linked to the main cluster and Lp1 ST42 isolates that were cocirculating at the time of the outbreak.

Surveillance of Australian Hajj pilgrims for carriage of potentially pathogenic bacteria: Data from two pilot studies.

AIM: To estimate the pharyngeal carriage rate of Neisseria meningitidis (N. meningitidis), Streptococcus pneumoniae (S. pneumoniae) and Staphylococcus aureus (S. aureus) among Australian Hajj pilgrims. METHODS: In 2014, surveillance was conducted in two phases among Australian Hajj pilgrims: The first phase during Hajj in Mina, and the second phase soon after returning home to Australia. Nasopharyngeal or oropharyngeal swabs were taken from participants then tested, firstly by nucleic acid testing, and also by standard culture. RESULTS: Of 183 participants recruited in the first phase, 26 (14.2%) tested positive for S. pneumoniae; 4 had received pneumococcal conjugate vaccine (PCV13). Only one tested positive for N. meningitidis (W). Of 93 2nd phase samples cultured, 17 (18.3%) grew S. aureus, all methicillin sensitive, 2 (2.2%) grew N. meningitidis (on subculture; one serotype B, one negative), and 1 (1%), from an unvaccinated pilgrim, grew S. pneumoniae. CONCLUSION: Relatively high carriage of S. pneumoniae and little meningococcal carriage was found. This indicates the importance of a larger study for improved infection surveillance and possible vaccine evaluation.

Treponema pallidum Strain Types and Association with Macrolide Resistance in Sydney, Australia: New TP0548 Gene Types Identified.

Strain typing of Treponema pallidum, using the three-target enhanced classification scheme, was performed with 191 samples obtained between 2004 and 2011 in Sydney, Australia. The most common strain type was 14d/g (92/191 samples [48%]). Two new TP0548 gene types were detected (m and n). Strain type was associated with macrolide resistance and possible acquisition outside Australia.

Treatment and outcomes of polymerase chain reaction-confirmed early syphilis.

UNLABELLED: Background Syphilis is resurgent among gay and bisexual men (GBM) despite effective treatment and widely available diagnostic serology. The polymerase chain reaction assay for Treponema pallidum (TP-PCR) is available, but little is known about the clinical features and outcomes for patients testing positive by TP-PCR. METHODS: Clinical data were collected from four medical practices for patients recording a positive TP-PCR result between 2004 and 2011. Demographic, serological, treatment and reinfection details were obtained. Results were stratified by HIV status and whether treatment conformed to international guidelines. RESULTS: 220 patients were positive for TP-PCR, of whom 92% were GBM. Seventeen (8.1%) were positive by TP-PCR before seroconversion. Almost one-third (32.1%) received treatment beyond that recommended in guidelines, and this was associated with HIV status (40.3% HIV positive vs 22.4% HIV negative, P<0.01). All but one patient with adequate follow up achieved serological cure. There was no significant difference in time to serological cure between the groups receiving standard therapy or enhanced therapy (95 vs 108 days; P=0.67) or between HIV positive and negative patients (93 vs 104 days, P=0.06). Nineteen patients were reinfected during follow up. CONCLUSION: TP-PCR aids early diagnosis of syphilis and may be reactive before conventional serological tests. Treatment outcomes for TP-PCR-positive early infection were excellent, but a significant proportion of patients received non-standard therapy. Expanded use of syphilis PCR testing in at-risk populations is recommended since early identification and treatment is likely to be important in controlling the current epidemic in GBM.

Clinical Utility of Urinary Cytology to Detect BK Viral Nephropathy.

BACKGROUND: Reactivation of BK polyoma virus can result in destructive viral allograft nephropathy (BKVAN) with limited treatment options. Screening programs using surrogate markers of viral replication are important preventive strategies, guiding immunosuppression reduction. METHODS: We prospectively evaluated the diagnostic test performance of urinary decoy cells and urinary SV40T immunochemistry of exfoliated cells, to screen for BKVAN, (defined by reference histology with SV40 immunohistochemistry, n = 704 samples), compared with quantitative viremia, from 211 kidney and 141 kidney-pancreas transplant recipients. RESULTS: The disease prevalence of BKVAN was 2.6%. Decoy cells occurred in 95 of 704 (13.5%) samples, with a sensitivity of 66.7%, specificity of 88.6%, positive predictive value (PPV) of 11.7%, and negative predictive value of 98.5% to predict histologically proven BKVAN. Quantification of decoy cells improved the PPV to 32.1% (10 ≥ cells threshold). Immunohistochemical staining of urinary exfoliated cells for SV40T improved sensitivity to 85.7%, detecting atypical or degenerate infected cells (specificity of 92.3% and PPV of 33.3%), but was hampered by technical failures. Viremia occurred in 90 of 704 (12.8%) with sensitivity of 96.3%, specificity of 90.3%, PPV of 31.5%, and negative predictive value of 99.8%. The receiver-operator curve performance of quantitative viremia surpassed decoy cells (area under the curve of 0.95 and 0.79, respectively, P = 0.0018 for differences). Combining decoy cell and BK viremia in a diagnostic matrix improved prediction of BKVAN and diagnostic risk stratification, especially for high-level positive results. CONCLUSIONS: Although quantified decoy cells are acceptable surrogate markers of BK viral replication with unexceptional test performances, quantitative viremia displayed superior test characteristics and is suggested as the screening test of choice.

Azithromycin-resistant syphilis-causing strains in Sydney, Australia: prevalence and risk factors.

Azithromycin has shown high efficacy in randomized trials when used for treating infectious syphilis in Africa. However, its use in clinical practice has been limited by the development of antimicrobial drug resistance. Resistance has not previously been reported from Australasia. The aim of this study was to determine the prevalence of and risk factors for azithromycin-resistant syphilis-causing strains in Sydney, Australia. We evaluated 409 samples that were PCR positive for Treponema pallidum DNA collected between 2004 and 2011 for the presence of the A2058G mutation, which confers resistance to macrolide antibiotics such as azithromycin. Overall, 84% of samples harbored the mutation. The prevalence of the mutation increased during the study period (P trend, 0.003). We also collected clinical and demographic data on 220 patients from whom these samples had been collected to determine factors associated with the A2058G mutation; 97% were from men who have sex with men. Reporting sex in countries other than Australia was associated with less macrolide resistance (adjusted odds ratio, 0.25; 95% confidence interval, 0.09 to 0.66; P = 0.005), with other study factors showing no association (age, HIV status, recent macrolide use, stage of syphilis, or history of prior syphilis). Azithromycin cannot be recommended as an alternative treatment for syphilis in Sydney.

Molecular characterizations of PCR-positive Mycoplasma pneumoniae specimens collected from Australia and China.

Mycoplasma pneumoniae is an important cause of community-acquired pneumonia (CAP). In this study, M. pneumoniae strains in PCR-positive specimens collected from patients in Sydney, Australia (30 samples), and Beijing, China (83 samples), were characterized using multilocus variable-number tandem-repeat (VNTR) analysis (MLVA), P1-restriction fragment length polymorphism (RFLP) analysis, and sequencing of domain V of the 23S rRNA gene to compare genotype distribution and macrolide resistance rates between locations. Eighteen distinct MLVA types were identified in specimens from Sydney, of which 10 were known (types E, G, J, M, N, P, U, V, S, and X) and 8 previously unknown. Strains were equally distributed between P1-RFLP type 1 and type 2 variants. Among samples from Beijing, MLVA types E, G, J, P, U, X, and Z and four new types were identified. Most specimens belonged to P1-RFLP type 1. A nomenclature based on five VNTR loci is proposed to designate MLVA patterns. Macrolide resistance-associated mutations were identified in only 1 of 30 specimens (3.3%) from Sydney and 71 of 83 (85.5%) from Beijing (P<0.05). This study demonstrated that although multiple individual M. pneumoniae strains were circulating in Beijing, the genotypes were less diverse than those in Sydney. However, the greatest regional difference was in the incidence of macrolide resistance, which may reflect differences in antibiotic use and/or measures in resistance control.

Failure of moxifloxacin treatment in Mycoplasma genitalium infections due to macrolide and fluoroquinolone resistance.

Increasing azithromycin treatment failure in sexually transmitted Mycoplasma genitalium infection, is linked to macrolide resistance and second-line treatment relies on the fluoroquinolone, moxifloxacin. We recently detected fluoroquinolone and macrolide resistance-associated mutations in 15% and 43%, respectively, of 143 initial M. genitalium PCR-positive specimens. For a subset of 33 Western Sydney Sexual Health Centre patients, clinical information and results of sequence analysis of M. genitalium macrolide and fluoroquinolone target genes - the 23S rRNA gene, and parC and gyrA, respectively - were used to examine whether mutations were associated with treatment failure. Macrolide resistance-associated mutations correlated with microbiological (p = 0.013) and clinical (p = 0.024) treatment failure, and fluoroquinolone resistance-associated mutations with microbiological moxifloxacin treatment failure (p = 0.005). We describe the first reported cases of clinical and microbiological moxifloxacin treatment failure. Failure of first- and second-line antibiotic treatment of M. genitalium infection is occurring and likely to increase with current treatment strategies.

Diagnosis of Barmah Forest virus infection by a nested real-time SYBR green RT-PCR assay.

Barmah Forest virus (BFV) is a mosquito borne (+) ssRNA alphavirus found only in Australia. It causes rash, myalgia and arthralgia in humans and is usually diagnosed serologically. We developed a real-time PCR assay to detect BFV in an effort to improve diagnosis early in the course of infection. The limit of detection was 16 genome equivalents with a specificity of 100%. Fifty five serum samples from BFV-infected patients were tested by the PCR. 52 of 53 antibody-positive samples were PCR negative. Two culture-positive (neutralizing antibody negative) samples were positive on first round PCR, while one sample (IgM and neutralizing antibody strongly positive, IgG negative) was positive on second round PCR, suggesting that viral RNA is detectable and transiently present in early infection. PCR can provide results faster than culture, is capable of high throughput and by sequencing the PCR product strain variants can be characterized.

Fluoroquinolone and macrolide resistance-associated mutations in Mycoplasma genitalium.

Mycoplasma genitalium is a significant sexually transmitted pathogen, causing up to 25% of cases of nongonococcal urethritis in men, and it is strongly associated with cervicitis and pelvic inflammatory disease in women. Currently, the usual first-line treatment is the macrolide antibiotic azithromycin, but an increasing incidence of treatment failure over the last 5 years suggests the emergence of antibiotic resistance. The mutations responsible for macrolide resistance have been found in the 23S rRNA gene in numerous M. genitalium populations. A second-line antibiotic, the fluoroquinolone moxifloxacin, was thought to be a reliable alternative when azithromycin began to fail, but recent studies have identified mutations that may confer fluoroquinolone resistance in the genes parC and gyrA. The aim of this study was to determine the prevalence of antibiotic resistance in M. genitalium in Sydney, Australia, by detecting relevant mutations in the 23S rRNA gene, parC, and gyrA. M. genitalium-positive DNA extracts of specimens, collected from patients attending sexual health clinics in Sydney, were tested by PCR amplification and DNA sequence alignment. The 186 specimens tested included 143 initial patient specimens and 43 second, or subsequent, specimens from 24 patients. We identified known macrolide resistance-associated mutations in the 23S rRNA gene in 43% of the initial patient samples and mutations potentially associated with fluoroquinolone resistance in parC or gyrA sequences in 15% of the initial patient samples. These findings support anecdotal clinical reports of azithromycin and moxifloxacin treatment failures in Sydney. Our results indicate that further surveillance is needed, and testing and treatment protocols for M. genitalium infections may need to be reviewed.