A novel murine model mimicking male genital Neisseria species infection using Neisseria musculi†.

With ~78 million cases yearly, the sexually transmitted bacterium Neisseria gonorrhoeae is an urgent threat to global public health due to continued emergence of antimicrobial resistance. In the male reproductive tract, untreated infections may cause permanent damage, poor sperm quality, and subsequently subfertility. Currently, few animal models exist for N. gonorrhoeae infection, which has strict human tropism, and available models have limited translatability to human disease. The absence of appropriate models inhibits the development of vital new diagnostics and treatments. However, the discovery of Neisseria musculi, a mouse oral cavity bacterium, offers much promise. This bacterium has already been used to develop an oral Neisseria infection model, but the feasibility of establishing urogenital gonococcal models is unexplored. We inoculated mice via the intrapenile route with N. musculi. We assessed bacterial burden throughout the male reproductive tract, the systemic and tissue-specific immune response 2-weeks postinfection, and the effect of infection on sperm health. Neisseria musculi was found in penis (2/5) and vas deferens (3/5) tissues. Infection altered immune cell counts: CD19+ (spleen, lymph node, penis), F4/80+ (spleen, lymph node, epididymus), and Gr1+ (penis) compared with noninfected mice. This culminated in sperm from infected mice having poor viability, motility, and morphology. We hypothesize that in the absence of testis infection, infection and inflammation in other reproductive is sufficient to damage sperm quality. Many results herein are consistent with outcomes of gonorrhoea infection, indicating the potential of this model as a tool for enhancing the understanding of Neisseria infections of the human male reproductive tract.

Application of the ViroKey® SQ FLEX assay for detection of cytomegalovirus antiviral resistance.

BACKGROUND: Cytomegalovirus (CMV) is a viral infection which establishes lifelong latency, often reactivating and causing disease in immunosuppressed individuals, including haematopoietic stem cell transplant (HSCT) recipients. Treatment can be problematic due to antiviral resistance which substantially increases the risk of patient mortality. Diagnostic testing capabilities for CMV antiviral resistance in Australia and elsewhere have traditionally relied on gene-specific Sanger sequencing approaches, however, are now being superseded by next generation sequencing protocols. OBJECTIVE: Provide a snapshot of local mutations and explore the feasibility of the ViroKeyࣨ® SQ FLEX Genotyping Assay (Vela Diagnostics Pty Ltd) by examining sequencing success. METHOD: Performed sequencing on adult (n = 38) and paediatric (n = 81) plasma samples, over a large range of viral loads (above and below the assay recommended threshold of ≥1,000 International Units (IU)/mL; noting most of our paediatric samples have loads <1,000 IU/mL). RESULTS: Eleven test runs (including three repeat runs; 14 to 15 samples per run) were conducted, and four runs were deemed valid. The overall individual sample success rate for the four evaluable test runs was 71.2% (42/59 samples); 80.4% (37/46) samples ≥1,000 IU/mL were valid. Ten clinically important antiviral resistance mutations were detected, the most common being A594V in the UL97 gene, found in 6 (5%) samples. CONCLUSIONS: A range of technical issues were experienced, however with improvement this platform could be a useful addition to routine pathology workflows, providing timely antiviral resistance results for patients undergoing HSCT.

Cytomegalovirus in children undergoing haematopoietic stem cell transplantation: a diagnostic and therapeutic approach to antiviral resistance.

Cytomegalovirus (CMV) is a ubiquitous virus which causes a mild illness in healthy individuals. In immunocompromised individuals, such as children receiving haematopoietic stem cell transplantation, CMV can reactivate, causing serious disease and increasing the risk of death. CMV can be effectively treated with antiviral drugs, but antiviral resistance is an increasingly common complication. Available therapies are associated with adverse effects such as bone marrow suppression and renal impairment, making the choice of appropriate treatment challenging. New agents are emerging and require evaluation in children to establish their role. This review will discuss established and emerging diagnostic tools and treatment options for CMV, including antiviral resistant CMV, in children undergoing haematopoietic stem cell transplant.

Mycoplasma hominis and Ureaplasma urealyticum infections in the immediate post-lung transplant period: A case series and literature review.

Mycoplasma hominis and Ureaplasma species infections in the post-transplant setting are believed to be donor-derived and can be associated with poor outcomes. Difficulty in culturing and identifying these organisms is a significant barrier to diagnosis and early intervention. Tetracyclines, macrolides and fluoroquinolones are the mainstay treatments to cure these infections; however, there are increasing reports of antibiotic resistance. In this case series, we report our single-centre experience with M. hominis and U. urealyticum infection after lung transplantation (9 recipients, all men, mean age 56 years). Delayed diagnosis was common. Young donor age (mean age 23 yrs) and high-risk donor social history (67%) were repeatedly noted in these cases, and all infections were associated with significant morbidity (anastomosis and sternal wound infection, empyema, mediastinitis, pericarditis). Two patients died; with one directly related to Ureaplasma urealyticum infection. In conclusion post lung transplant M. hominis, and U. urealyticum infections are challenging and carry high morbidity. More prospective studies are required to assess the true prevalence, full spectrum of complications and utility of molecular diagnostics to aid early diagnosis and identify antibiotic susceptibility of Mycoplasma and Ureaplasma infections in the post-lung transplant setting.

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.

Primary oral vaccination followed by a vaginal pull protects mice against genital HSV-2 infection.

PROBLEM: HSV-2 infected more than 491 million people aged 15-49 world-wide in 2016. The morbidity associated with recurrent infections and the increased risk of HIV infection make this a major health problem. To date there is no effective vaccine. Because HSV-2 ascends to the dorsal route ganglion within 12-18 h of infection, an effective vaccine will need to elicit a strong local resident CD8+ T cell response to prevent the infection from becoming life-long. METHOD OF STUDY: Using a mouse model we investigated the potential of oral immunization with a novel lipid adjuvant (LiporaleTM ) followed by local vaginal application of an inflammatory agents to protect against primary HSV-2 infections. RESULTS: Oral vaccination of mice with live-attenuated HSV-2 in Liporale followed by vaginal application of DNFB or CXCL9/10 led to recruitment of tissue-resident CD8+ memory cells into the genital epithelia. This prime and pull vaccination strategy provided complete protection against wild-type HSV-2 challenge and prevented viral dissemination to the spinal cords. CONCLUSIONS: Activation of mucosal immunity by oral immunization, combined with induction of transient local genital inflammation can recruit long-lived tissue resident CD8+ T cells into the genital epithelium, providing significant protection against primary HSV-2 infection.

Two Treponema pallidum strains account for the majority of syphilis infections, including among females, in Queensland, Australia.

Abstract: An ongoing outbreak of syphilis in Australia, first reported in the state of Queensland in 2011, has led to increasing cases of congenital syphilis, including several deaths. Here, we applied multi-locus sequence typing (MLST) on available Treponema pallidum PCR-positive samples from the state of Queensland from the beginning of the outbreak to July 2020. In total, 393 samples from 337 males and 56 females were genotyped. Of 36 different Treponema pallidum sequence types (ST) observed, the two most common STs, ST 1 (also reported to be a dominant strain in various other countries) and ST 100 (the latter differing from ST 1 by only one single nucleotide polymorphism (SNP) based on the MLST scheme), together comprised 69% (271/393) of all samples, including the majority of samples in females (79%; 44/56). ST 1 was prevalent throughout the entire study period. Both strains remained the most common STs during the year 2020 where social distancing and other measures were implemented due to the COVID-19 pandemic. Both STs had high male-to-female ratios and included male rectal infections, therefore suggestive of occurrence primarily among men-who-have-sex-with-men (MSM). Hence, bridging from MSM to heterosexual networks may potentially contribute to infections among females, but further studies are needed to confirm this. Overall, there was considerable diversity of Treponema pallidum genotypes observed throughout the study period, but the fact that two key strains accounted for the majority of infections, including among females, stresses the need for further investigations into the transmission of these strains, and potentially a need for targeted public health interventions to better control the spread of syphilis in Queensland.

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.

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.

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.

The Prevalence of Antimicrobial Resistant Neisseria gonorrhoeae in Papua New Guinea: A Systematic Review and Meta-Analysis.

Neisseria gonorrhoeae antimicrobial resistance (NG AMR) has become an urgent concern globally. The World Health Organization, the United States of America Centers for Disease Control, and other regulators have called to improve resistance-testing methods to enhance NG AMR surveillance. NG AMR surveillance remains critical in informing treatment; unfortunately, this is often lacking in settings with limited resources, such as Papua New Guinea (PNG). We conducted a systematic review and a prevalence meta-analysis, and provided an overview of NG AMR in PNG. We showed the lack of NG AMR data in the last decade, and emphasized the need for NG AMR surveillance in PNG. Since NG AMR testing by the NG culture method is unreliable in PNG, we suggested using molecular tests to complement and enhance NG AMR surveillance.

Global phylogeny of Treponema pallidum lineages reveals recent expansion and spread of contemporary syphilis.

Syphilis, which is caused by the sexually transmitted bacterium Treponema pallidum subsp. pallidum, has an estimated 6.3 million cases worldwide per annum. In the past ten years, the incidence of syphilis has increased by more than 150% in some high-income countries, but the evolution and epidemiology of the epidemic are poorly understood. To characterize the global population structure of T. pallidum, we assembled a geographically and temporally diverse collection of 726 genomes from 626 clinical and 100 laboratory samples collected in 23 countries. We applied phylogenetic analyses and clustering, and found that the global syphilis population comprises just two deeply branching lineages, Nichols and SS14. Both lineages are currently circulating in 12 of the 23 countries sampled. We subdivided T. p. pallidum into 17 distinct sublineages to provide further phylodynamic resolution. Importantly, two Nichols sublineages have expanded clonally across 9 countries contemporaneously with SS14. Moreover, pairwise genome analyses revealed examples of isolates collected within the last 20 years from 14 different countries that had genetically identical core genomes, which might indicate frequent exchange through international transmission. It is striking that most samples collected before 1983 are phylogenetically distinct from more recently isolated sublineages. Using Bayesian temporal analysis, we detected a population bottleneck occurring during the late 1990s, followed by rapid population expansion in the 2000s that was driven by the dominant T. pallidum sublineages circulating today. This expansion may be linked to changing epidemiology, immune evasion or fitness under antimicrobial selection pressure, since many of the contemporary syphilis lineages we have characterized are resistant to macrolides.

Impairments in glycaemic control do not increase linearly with repeated nights of sleep restriction in healthy adults: a randomised controlled trial.

Evidence suggests reduced glycaemic control following sleep restriction in healthy individuals. However, it remains unknown if impairments in glycaemic control increase with each additional night of sleep restriction in a linear manner. This randomised crossover study aimed to determine if the impairment in glycaemic control increases with each additional night of sleep restriction. Ten healthy individuals underwent 4 nights of control sleep (8 hours in bed) and 4 nights of sleep restriction (4 hours in bed) in a sleep laboratory. An oral glucose tolerance test was conducted each morning. Serum glucose and insulin were measured. Glucose and insulin area under the curve were higher overall in the sleep restriction trial compared with control (p < 0.001 and p = 0.033); however, no effect of day (p = 0.620 and p = 0.863) or interaction effect (p = 0.152 and p = 0.285) were observed. This supports previous literature showing a detrimental impact of sleep restriction on glucose regulation. The present findings, however, suggest the impairment in glycaemic control does not increase in a linear manner with an increasing number of nights of sleep restriction. This may have implications for the design of future studies examining sleep restriction and glycaemic control. Novelty: Four nights of sleep restriction impaired glycaemic control in healthy individuals, but did not do so in a linear manner. No effect of number of nights of restriction was found for glucose or insulin, which may have implications for future studies.

Mycoplasma genitalium infections can comprise a mixture of both fluoroquinolone-susceptible and fluoroquinolone-resistant strains.

BACKGROUND: Mycoplasma genitalium was recently added to the CDC's antimicrobial resistance threats 'watch list', as it has rapidly become resistant to mainstay treatments. In Australia, treatment failure with fluoroquinolones remain commonplace, even when Sanger sequencing fails to identify evidence of resistance mutations. METHODS: Suspecting that Sanger sequencing may miss low-load mixed infections, we applied three additional PCR-based approaches (allele-specific primer-based PCR, probe-based PCR and amplicon deep sequencing) to detect mutations associated with fluoroquinolone susceptibility/resistance. We focused on resistance mutations at amino acid positions 83 and 87 of parC, as these were previously shown to be common in Australia. RESULTS: Our results showed evidence of mixtures of fluoroquinolone-susceptible and -resistant strains in up to 27/423 samples (6.4%). These included 1 sample that was indicated to be mixed by Sanger sequencing and all three additional PCR methods, 6 samples detected by two or more of the additional PCRs but not by Sanger sequencing and finally 20 samples that were detected by only one of the additional PCR methods. A key question was whether Sanger sequencing failed to detect fluoroquinolone resistance in any samples; overall, we observed that Sanger sequencing failed to detect fluoroquinolone resistance in up to 3.8% (16/423) of samples. CONCLUSIONS: The presence of mixed susceptibility infections may have important implications for clinical patient management and stresses the need for appropriate detection of resistance and selection of antimicrobials to ensure appropriate treatment of M. genitalium infections.