DNA methylation at individual CpG-sites of EPB41L3, HTERT and FAM19A4 are useful for detection of cervical high-grade squamous intraepithelial lesions (HSIL) or worse: Analysis of individual CpG-sites outperforms averaging.

Global methylation analysis of gene promoters is promising for detection of high-grade squamous intraepithelial lesions or worse (HSIL+) in high-risk human papillomavirus (hrHPV)-positive women. However, diagnostic performance of methylation data at individual CpG-sites is limited. We explored methylation for predicting HSIL+ in self- and clinician-collected samples from Papua New Guinea. Methylation of EPB41L3 (1-6 CpG-sites), hTERT (1-10 CpG-sites) and FAM19A4 (1-5 CpG-sites) was assessed through pyrosequencing from 44 HPV+ samples (4 cancers, 19 HSIL, 4 low-grade squamous intraepithelial lesions (LSIL), 17 normal). New primers were designed for FAM19A4 directed to the first exon region not explored previously. In clinician-collected samples, methylation at CpG-sites 4 and 5 of EPB41L3 were the best HSIL predictors (AUC >0.83) and CpG-site 4 for cancer (0.925). Combination of EPB41L3 sites 2/4 plus FAM19A4 site 1 were the best HSIL+ markers [100% sensitivity, 63.2% specificity]. Methylation at CpG-site 5 of FAM19A4 was the best HSIL predictor (0.67) in self-collected samples, and CpG-sites 1 and 3 of FAM19A4 for cancer (0.77). Combined, FAM19A4 site 1 plus HPV 16/18 detection yielded sensitivity of 82.6% and specificity of 61.9%. In conclusion, methylation at individual CpG-sites of EPB41L3 and FAM19A4 outperformed global analysis and improved HSIL+ detection, warranting further investigation.

Performance of CADM1, MAL and miR124-2 methylation as triage markers for early detection of cervical cancer in self-collected and clinician-collected samples: an exploratory observational study in Papua New Guinea.

OBJECTIVE: WHO recommends human papillomavirus (HPV) testing for cervical screening, with triage of high-risk HPV (hrHPV) positive women. However, there are limitations to effective triage for low-resource, high-burden settings, such as Papua New Guinea. In this exploratory study, we assessed the performance of host methylation as triage tools for predicting high-grade squamous intraepithelial lesions (HSIL) in self-collected and clinician-collected samples. DESIGN: Exploratory observational study. SETTING: Provincial hospital, same-day cervical screen-and-treat trial, Papua New Guinea. PARTICIPANTS: 44 hrHPV+women, with paired self/clinician-collected samples (4 squamous cell carcinomas (SCC), 19 HSIL, 4 low-grade squamous intraepithelial lesions, 17 normal). PRIMARY AND SECONDARY OUTCOME MEASURES: Methylation levels of CADM1, MAL and miR124-2 analysed by methylation-specific PCRs against the clinical endpoint of HSIL or SCC (HSIL+) measured using liquid-based-cytology/p16-Ki67 stain. RESULTS: In clinician-collected samples, MAL and miR124-2 methylation levels were significantly higher with increasing grade of disease (p=0.0046 and p<0.0015, respectively). miR124-2 was the best predictor of HSIL (area under the curve, AUC 0.819) while MAL of SCC (AUC 0.856). In self-collected samples, MAL best predicted HSIL (AUC 0.595) while miR124-2 SCC (AUC 0.812). Combined miR124-2/MAL methylation yielded sensitivity and specificity for HSIL+ of 90.5% (95% CI 69.6% to 98.8%) and 70% (95% CI 45.7% to 88.1%), respectively, in clinician-collected samples, and 81.8% (95% CI 59.7% to 94.8%) and 47.6% (95% CI 25.7% to 70.2%), respectively, in self-collected samples. miR124-2/MAL plus HPV16/HPV18 improved sensitivity for HSIL+ (95.2%, 95% CI 76.2% to 99.9%) but decreased specificity (55.0%, 95% CI 31.5% to 76.9%). CONCLUSION: miR124-2/MAL methylation is a potential triage strategy for the detection of HSIL/SCC in low-income and middle-income country.

Vaginal anaerobes are associated with cervicitis: A case-control study.

OBJECTIVES: Cervicitis is associated with important reproductive sequelae. Primary causes include chlamydia and gonorrhoea, but a known sexually transmitted infection (STI) is not identified in >50% of cases (i.e. STI-negative cervicitis). Bacterial vaginosis (BV) and specific BV-associated bacteria have also been associated with cervicitis, but data are limited. We investigated the association between STI-negative cervicitis and vaginal microbiota composition. METHODS: This was a case-control sub-study of the OhMG study conducted at the Melbourne Sexual Health Centre. Cases were women with cervicitis who tested negative for STIs (STI-negative cervicitis, n = 64). Controls were STI-negative asymptomatic women attending for STI-screening (n = 128). The vaginal microbiota was characterised using 16S rRNA gene sequencing. Vaginal community state types were compared between cases and controls using logistic regression. Differential abundance analysis was performed to identify taxa associated with STI-negative cervicitis. RESULTS: STI-negative cervicitis cases were more likely than controls to have a Lactobacillus-deficient non-optimal microbiota (adjusted-odds-ratio 2.55, 95% CI 1.18-5.50). Compared to controls, cases had increased abundance of four BV-associated bacteria (Gardnerella, Fannyhessea vaginae, Prevotella bivia, Dialister micraerophilus) and decreased abundance of optimal lactobacilli. CONCLUSIONS: We report a positive association between non-optimal vaginal microbiota composition and STI-negative cervicitis. Specific anaerobic BV-associated bacteria may represent infectious causes of cervicitis.

Lactate metabolism promotes in vivo fitness during Acinetobacter baumannii infection.

Acinetobacter baumannii is one of the most prevalent causes of nosocomial infections worldwide. However, a paucity of information exists regarding the connection between metabolic capacity and in vivo bacterial fitness. Elevated lactate is a key marker of severe sepsis. We have previously shown that the putative A. baumannii lactate permease gene, lldP, is upregulated during in vivo infection. Here, we confirm that lldP expression is upregulated in three A. baumannii strains during a mammalian systemic infection. Utilising a transposon mutant disrupted for lldP in the contemporary clinical strain AB5075-UW, and a complemented strain, we confirmed its role in the in vitro utilisation of l-(+)-lactate. Furthermore, disruption of the lactate metabolism pathway resulted in reduced bacterial fitness during an in vivo systemic murine competition assay. The disruption of lldP had no impact on the susceptibility of this strain to complement mediated killing by healthy human serum. However, growth in biologically relevant concentrations of lactate observed during severe sepsis, led to bacterial tolerance to killing by healthy human blood, a phenotype that was abolished in the lldP mutant. This study highlights the importance of the lactate metabolism pathway for survival and growth of A. baumannii during infection.

Human papillomavirus negative high grade cervical lesions and cancers: Suggested guidance for HPV testing quality assurance.

BACKGROUND: Some high-grade cervical lesions and cervical cancers (HSIL+) test negative for human papillomavirus (HPV). The HPV-negative fraction varies between 0.03 % and 15 % between different laboratories. Monitoring and extended re-analysis of HPV-negative HSIL+ could thus be helpful to monitor performance of HPV testing services. We aimed to a) provide a real-life example of a quality assurance (QA) program based on re-analysis of HPV-negative HSIL+ and b) develop international guidance for QA of HPV testing services based on standardized identification of apparently HPV-negative HSIL+ and extended re-analysis, either by the primary laboratory or by a national HPV reference laboratory (NRL). METHODS: There were 116 initially HPV-negative cervical specimens (31 histopathology specimens and 85 liquid-based cytology samples) sent to the Swedish HPV Reference Laboratory for re-testing. Based on the results, an international QA guidance was developed through an iterative consensus process. RESULT: Standard PCR testing detected HPV in 55.2 % (64/116) of initially "HPV-negative" samples. Whole genome sequencing of PCR-negative samples identified HPV in an additional 7 samples (overall 61.2 % HPV positivity). Reasons for failure to detect HPV in an HSIL+ lesion are listed and guidance to identify cases for extended re-testing, including which information should be included when referring samples to an NRL are presented. CONCLUSION: Monitoring the proportion of and reasons for failure to detect HPV in HSIL+ will help support high performance and quality improvement of HPV testing services. We encourage implementation of QA strategies based on re-analysis of "HPV negative" HSIL+ samples.

Evaluation of TIB Molbiol LightMix® assays for detection of Mycoplasma genitalium and key resistance mutations for macrolides and fluoroquinolones.

The LightMix® Modular Mycoplasma Macrolide and LightMix® Modular parC Fluoroquinolone Resistance assays (TIB Molbiol) were evaluated using sequential Mycoplasma genitalium positive (n = 125) and negative (n = 93) clinical samples. Results were compared to the results of an established commercial assay (ResistancePlus MG assay, SpeeDx Pty Ltd) or Sanger sequencing (for parC). Detection of M. genitalium by the TIB Molbiol assay had a high agreement with the reference assay, with a positive percent agreement (PPA) of 97.6 [95% confidence interval (CI): 93.1-99.5] and negative percent agreement (NPA) of 95.7 (95% CI: 89.5-98.8). From 105 positive samples, macrolide resistance detection had a PPA of 100% (95% CI: 93.7-100) and NPA of 81.3% (95% CI: 67.4-91.1). For the detection of fluroquinolone resistance mutation G248T/S83I or "other mutation" in the quinolone resistance determinant region, from 95 samples there was 100% (95% CI: 86.3-100) sensitivity and 100% (95% CI: 94.5-100) specificity. The understanding of the basis for fluoroquinolone treatment failure is still developing; it is therefore important to use the output of parC-based resistance assays with caution to avoid the inappropriate use of antibiotic therapies, especially considering the limited number of alternative treatments.

Prevotella and Gardnerella Are Associated With Treatment Failure Following First-line Antibiotics for Bacterial Vaginosis.

BACKGROUND: Bacterial vaginosis (BV) is a common vaginal dysbiosis that often recurs following first-line antibiotics. We investigated if vaginal microbiota composition was associated with BV recurrence. METHODS: We analyzed samples and data from 121 women who participated in 3 published trials evaluating novel interventions for improving BV cure, including concurrent antibiotic treatment of regular sexual partners (RSPs). Women diagnosed with BV received first-line antibiotics and self-collected vaginal swabs pretreatment and the day after finishing antibiotics (immediately posttreatment). 16S rRNA gene sequencing was performed on vaginal samples. Logistic regression explored associations between BV recurrence and features of the vaginal microbiota pre- and posttreatment. RESULTS: Sixteen women (13% [95% confidence interval {CI}, 8%-21%]) experienced BV recurrence within 1 month of treatment. Women with an untreated RSP were more likely to experience recurrence than women with no RSP (P = .008) or an RSP who received treatment (P = .011). A higher abundance of Prevotella pretreatment (adjusted odds ratio [AOR], 1.35 [95% CI, 1.05-1.91]) and Gardnerella immediately posttreatment (AOR, 1.23 [95% CI, 1.03-1.49]) were associated with increased odds of BV recurrence. CONCLUSIONS: Having specific Prevotella spp prior to recommended treatment and persistence of Gardnerella immediately posttreatment may contribute to the high rates of BV recurrence. Interventions that target these taxa are likely required to achieve sustained BV cure.

Quantitation of Mycoplasma genitalium using droplet digital PCR.

Mycoplasma genitalium is a sexually transmitted infection with increasing concerns around antimicrobial resistance. Droplet digital PCR (ddPCR) is a rapid quantification method with high precision that may be useful for absolute quantitation of bacteria in samples. This study aimed to develop a ddPCR assay for the quantification of M. genitalium. ddPCR targeting the gene mgpB was established and analysed using the QX100 ddPCR system. The assay was evaluated against quantitated DNA standards, and then in comparison to an established quantitative PCR performed on the Lightcycler 480 II. DNA template of increasing complexity was used, including synthetic double stranded DNA, DNA extracts from laboratory-cultured M. genitalium strains (n = 17) and DNA from M. genitalium-positive clinical samples (n = 21). There was a strong correlation between ddPCR concentration estimates and measured DNA standards (r2 = 0.997), and between ddPCR and qPCR quantitation for different templates (r2 ranging from 0.953 to 0.997). ddPCR reliably detected template in a range from <10 copies per reaction to >104 copies per reaction and demonstrated linearity over dilution series. Concentration estimates by ddPCR were reproducibly less than those determine by qPCR. ddPCR demonstrated precise and reproducible quantitation of M. genitalium with a variety of templates.

gyrA Mutations in Mycoplasma genitalium and Their Contribution to Moxifloxacin Failure: Time for the Next Generation of Resistance-Guided Therapy.

BACKGROUND: Although single nucleotide polymorphisms (SNPs) in Mycoplasma genitalium parC contribute to fluoroquinolone treatment failure, data are limited for the homologous gene, gyrA. This study investigated the prevalence of gyrA SNPs and their contribution to fluoroquinolone failure. METHODS: Samples from 411 patients (male and female) undergoing treatment for M. genitalium infection (Melbourne Sexual Health Centre, March 2019-February 2020) were analyzed by Sanger sequencing (gyrA and parC). For patients treated with moxifloxacin (n = 194), the association between SNPs and microbiologic treatment outcome was analyzed. RESULTS: The most common parC SNP was G248T/S83I (21.1% of samples), followed by D87N (2.3%). The most common gyrA SNP was G285A/M95I (7.1%). Dual parC/gyrA SNPs were found in 8.6% of cases. One third of infections harboring parC G248T/S83I SNP had a concurrent SNP in gyrA conferring M95I. SNPs in gyrA cooccurred with parC S83I variations. Treatment failure was higher in patients with parC S83I/gyrA dual SNPs when compared with infections with single S83I SNP alone from analysis of (1) 194 cases in this study (81.2% vs 45.8%, P = .047), and (2) pooled analysis of a larger population of 535 cases (80.6% vs 43.2%; P = .0027), indicating a strong additive effect. CONCLUSIONS: Compared with parC S83I SNP alone, M. genitalium infections with dual mutations affecting parC/gyrA had twice the likelihood of failing moxifloxacin. Although antimicrobial resistance varies by region globally, these data indicate that gyrA should be considered as a target for future resistance assays in Australasia. We propose a strategy for the next generation of resistance-guided therapy incorporating parC and gyrA testing.

Performance of Human Papillomavirus Attribution Algorithms to Predict Causative Genotypes in Anal High-Grade Lesions.

BACKGROUND: Gay and bisexual men (GBM) are at increased risk of human papillomavirus (HPV)-associated anal high-grade squamous intraepithelial lesions (HSILs). Understanding the fractions of HSILs attributable to HPV genotypes is important to inform potential impacts of screening and vaccination strategies. However, multiple infections are common, making attribution of causative types difficult. Algorithms developed for predicting HSIL-causative genotype fractions have never been compared with a reference standard in GBM. METHOD: Samples were from the Study of the Prevention of Anal Cancer. Baseline HPV genotypes detected in anal swab samples (160 participants) were compared with HPV genotypes in anal HSILs (222 lesions) determined by laser capture microdissection (LCM). Five algorithms were compared: proportional, hierarchical, maximum, minimum, and maximum likelihood estimation. RESULTS: All algorithms predicted HPV-16 as the most common HSIL-causative genotype, and proportions differed from LCM detection (37.8%) by algorithm (with differences of -6.1%, +20.9%, -20.4%, +2.9%, and +2.2% respectively). Fractions predicted using the proportional method showed a strong positive correlation with LCM, overall (R = 0.73 and P = .002), and by human immunodeficiency virus (HIV) status (HIV positive, R = 0.74 and P = .001; HIV-negative, R = 0.68 and P = .005). CONCLUSIONS: Algorithms produced a range of inaccurate estimates of HSIL attribution, with the proportional algorithm performing best. The high occurrence of multiple HPV infections means that these algorithms may be of limited use in GBM.

Comparison of Seegene AnyPlexTM II STI-7e with standard-of-care diagnostic methods for the detection of Mycoplasma genitalium, Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis.

The AnyPlexTM II STI-7e panel assay (Seegene) detects seven sexually transmitted organisms (Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, M. hominis, Ureaplasma urealyticum, U. parvum, and Trichomonas vaginalis). This study compared the performance of AnyPlexTM II STI-7e with standard-of-care diagnostic methods. Samples (cervical or vaginal swabs, or urine) from 1330 women were tested on standard-of-care assays; 83/1318 (6.3%) tested positive for M. genitalium (ResistancePlus® MG), 99/1317 (7.5%) positive for C. trachomatis and 11/1316 (0.8%) positive for N. gonorrhoeae (Hologic® Aptima Combo 2®), and 6/689 (0.9%) positive for T. vaginalis (wet mount microscopy). AnyPlexTM II STI-7e had good agreement for the detection of M. genitalium [Cohen's kappa of 0.80, 95% confidence intervals (CI) 0.74-0.87] and C. trachomatis (kappa of 0.87, 95% CI 0.82-0.92), with positive and negative % agreement >96% for both infections. There was lower agreement for the detection of N. gonorrhoeae (kappa of 0.37, 95%CI 0.19-0.55) and T. vaginalis (kappa of 0.521, 95%CI 0.25-0.80). In summary, the test performed well in this comparison for M. genitalium and C. trachomatis detection, but results were less conclusive for N. gonorrhoeae and T. vaginalis due to low prevalence in the population.

The Urethral Microbiota of Men with and without Idiopathic Urethritis.

Nongonococcal urethritis (NGU) is a common genital tract syndrome in men, and up to 50% of cases are considered idiopathic, i.e., no etiological agent is identified. This poses challenges for clinicians in the diagnosis and treatment of NGU and often results in antibiotic misuse and overuse. Therefore, to identify potential infectious causes of urethritis and inform clinical management of urethritis cases, we characterized and compared the urethral microbiota of men with and without idiopathic urethritis. Participants were derived from a case-control study that examined viral and bacterial pathogens and sexual practices associated with NGU. Men with NGU who tested negative for established causes of NGU (Chlamydia trachomatis, Mycoplasma genitalium, Trichomonas vaginalis, adenoviruses, herpes simplex virus [HSV]-1, and/or HSV-2) were classified as idiopathic cases, and the controls were men reporting no current urethral symptoms. Men provided a urine sample that was used to characterize the urethral microbiota using 16S rRNA gene sequencing. Bacterial taxa associated with idiopathic urethritis were identified using analysis of compositions of microbiomes with bias correction. When stratified by sex of sexual partner, we found that the abundance of Haemophilus influenzae was significantly increased in men who have sex with men with idiopathic urethritis, and the abundance of Corynebacterium was significantly increased in men who have sex with women with idiopathic urethritis. Other taxa, including Ureaplasma, Staphylococcus haemolyticus, Streptococcus pyogenes, Escherichia, and Streptococcus pneumoniae/pseudopneumoniae, dominated the urethral microbiota of idiopathic urethritis cases but not controls, suggesting that these organisms may also contribute to urethritis. Importantly, the taxa we identified represent biologically plausible causes of urethritis and should be prioritized for future study. IMPORTANCE Nongonococcal urethritis (NGU) is the commonest genital tract syndrome in men and is nearly universally presumptively treated with an antibiotic. Common causes of NGU include Chlamydia trachomatis and Mycoplasma genitalium, but in more than 50% of cases, an infectious cause is not identified. In this case-control study, we found that the urethral microbiota composition differed between men with and without idiopathic urethritis and differed by sex of sexual partner. We identified specific bacterial taxa that were associated with idiopathic urethritis, including Haemophilus influenzae and Corynebacterium. These data, together with the finding that key bacterial taxa were found to dominate the urethral microbiota of cases but not controls, suggest that a range of bacteria contribute to urethritis and that these organisms may be influenced by sexual practices. Through identifying the infectious causes of urethritis, we can inform appropriate targeted diagnostic and treatment practices and importantly reduce misuse and overuse of antibiotics.

Mycoplasma genitalium: enhanced management using expanded resistance-guided treatment strategies.

Mycoplasma genitalium is an emerging sexually transmitted bacterium that is gaining attention because of the impact escalating antimicrobial resistance (AMR) is having on patient management. Of additional concern is that increased availability of testing appears to be resulting in screening practices that are not supported by clinical guidelines. This results in increasing numbers of asymptomatic M. genitalium infections being identified, which when combined with AMR issues, creates significant challenges for patients and clinicians. Rapidly rising levels of AMR, coupled with limited alternative treatment options, means patients can enter cycles of complex antimicrobial regimens that may cause more harm than the infection itself. In this review, we discuss the emergence of AMR and the implication for treatment practices, highlight the recommendations for testing but not screening for M. genitalium , and discuss expansion of individualised treatment strategies, to curb the emergence of resistance and improve outcomes for patients. We also provide suggestions for future research on the transmission and spread of resistance, to enhance global surveillance of this antimicrobial resistant pathogen and inform the revision of local and international treatment strategies.

parC Variants in Mycoplasma genitalium: Trends over Time and Association with Moxifloxacin Failure.

Prevalence, trends, and treatment outcome estimates were generated for parC variants in macrolide-resistant Mycoplasma genitalium. Among 539 cases, the most common amino acid change was S83I, which increased from 13% in 2012 to 2013, to 23% in 2019 to 2020 (Ptrend = 0.046). From 381 moxifloxacin treatments, failure occurred in 58.7% (95% confidence interval [CI], 46.7 to 69.9) of cases with S83I. Other changes affecting S83 or D87 were uncommon and minor contributors to failure. The absence of S83I was highly predictive of moxifloxacin cure (96.4%; 95% CI, 93.7 to 98.2), highlighting diagnostic potential.

Impact of 16S rRNA Single Nucleotide Polymorphisms on Mycoplasma genitalium Organism Load with Doxycycline Treatment.

Doxycycline targets the 16S rRNA and is widely used for the treatment of sexually transmitted infections. While it is not highly effective at eradicating Mycoplasma genitalium infections, it can reduce organism load. The aim of this study was to investigate the association between single nucleotide polymorphisms (SNPs) in the 16S rRNA gene of M. genitalium and change in organism load. M. genitalium samples were collected from 56 men prior to commencing doxycycline and at a median of 13 of 14 doses. These were sequenced for the 16S rRNA, and the association between 16S rRNA SNPs and change in organism load was determined. 16S rRNA sequences were available for 52/56 (92.9%) M. genitalium-infected men, of which 20 (38.5%) had an undetectable load, 26 (50.0%) had a decrease in M. genitalium load (median change of 105-fold), and 6 (11.5%) had an increase in load (median change of 5-fold). The most common SNPs identified were A742G (10/52 [19.2%]), GG960-961TT/C (7/52 [13.5%]), and C1435T (28/52 [53.8%]) (M. genitalium numbering). None were associated with a change in organism load (P = 0.76, 0.16, and 0.98, respectively). Using pooled published data from 28 isolates, no clear relationship between the SNPs and doxycycline MIC was identified. In conclusion, the low efficacy of doxycycline against M. genitalium does not appear to be due to variation in the 16S rRNA gene.

Novel probe-based melting curve assays for the characterization of fluoroquinolone resistance in Mycoplasma genitalium.

BACKGROUND: Mycoplasma genitalium infection is a sexually transmitted infection that has rapidly become resistant to mainstay treatments. While individualized treatment approaches have been recommended and adopted for macrolides, individualized therapy for fluoroquinolones has not yet been explored, due to a lack of commercial molecular assays and a lack of confidence in specific mutations associated with resistance. In another recent study, we defined a clear role and diagnostic utility in focusing on the absence of resistance mutations to inform microbial cure with fluoroquinolone antimicrobials. METHODS: We developed two proof-of-concept molecular tests that focus on detection of M. genitalium and characterization of WT parC sequences that are strongly linked to fluoroquinolone susceptibility. RESULTS: We screened a total of 227 M. genitalium-positive samples using novel molecular beacon and dual hybridization probe assays. These assays were able to detect M. genitalium and characterize fluoroquinolone susceptibility in 143/227 (63%) samples, based on clear differences in melting peak temperatures. The results of these molecular assays were in 100% agreement with 'gold standard' Sanger sequencing. Additionally, WT parC sequences were readily distinguished from M. genitalium samples harbouring parC mutations of known or suspected clinical significance. The ability of the assays to successfully characterize fluoroquinolone susceptibility and resistance was reduced in low M. genitalium load samples. CONCLUSIONS: These proof-of-concept assays have considerable potential to improve individualized treatment approaches and rationalize tests of cure for M. genitalium infection. The ability to initiate individualized treatment in up to two-thirds of cases will enhance antimicrobial stewardship for this challenging pathogen.

Individualised treatment of Mycoplasma genitalium infection-incorporation of fluoroquinolone resistance testing into clinical care.

Mycoplasma genitalium is an emerging global health threat, due to an alarming rise in antimicrobial resistance. Although individualised treatment approaches have been successfully adopted for macrolides, treatment is complicated by rising rates of fluoroquinolone resistance and by the scarcity of alternative treatment options. In this Personal View, we discuss the available data within the literature and highlight issues surrounding individualised treatment using fluoroquinolones, including the hesitation to focus on inclusion of ParC fluoroquinolone resistance mutations for guiding antimicrobial treatments. We propose that there is a clear role for diagnostics that focus on the absence of resistance mutations (ie, wild-type sequences and antimicrobial susceptibility) to inform microbial cure following fluoroquinolone antimicrobials, with Australian data strongly supporting this approach. The development of molecular tests that incorporate markers to detect both wild-type and only the most common ParC mutation, Ser83Ile, could greatly improve first-line antimicrobial selection and stewardship, individualise tests of cure, and be extremely useful in the care of patients with M genitalium infection.

Random amplified polymorphic DNA analysis reveals no clear link between Staphylococcus epidermidis and acute mastitis.

Mastitis is commonly experienced by breastfeeding women. While Staphylococcus aureus is usually implicated in infectious mastitis, coagulase-negative staphylococci (CoNS) are a possible alternative pathogen. This case-control study examined the role of CoNS in mastitis using isolates cultured from breast milk of 20 women with mastitis and 16 women without mastitis. Gene sequencing determined bacterial species, and random amplified polymorphic DNA (RAPD) analysis investigated strain-level variation. The majority of CoNS isolates were Staphylococcus epidermidis (182/199; 91%). RAPD analysis identified 33 unique S. epidermidis profiles, with no specific profile associated with mastitis cases.