Maximizing the Neisseria gonorrhoeae confirmatory rate and the genotypic detection of ciprofloxacin resistance for samples screened with cobas CT/NG.

Supplementary nucleic acid amplification testing for Neisseria gonorrhoeae (NG) is widely used to circumvent specificity problems associated with extragenital sites. Here, we compared different supplementary approaches for confirming NG-positive samples from the cobas 4800 CT/NG (c4800) and cobas 6800 CT/NG (c6800) assays using the ResistancePlusGC (RP-GC) assay, which in addition to detecting NG, also predicts ciprofloxacin susceptibility via NG gyrA characterization. Two different nucleic acid extraction techniques were investigated for RP-GC detection; extracts from c4800 (c4800-RP-GC) and MagNA Pure 96 (MP96-RP-GC). NG-positive (n = 300) and -negative (n = 150) samples in cobas PCR media from routine c4800 testing were retrospectively retested with c4800, c6800, c4800-RP-GC, and MP96-RP-GC. Selected samples were also tested with Xpert CT/NG (Xpert) for discrepant analysis. The gyrA status was compared to ETEST ciprofloxacin susceptibility or non-susceptibility for recovered isolates (n = 63). Extragenital confirmatory rates were higher for MP96-RP-GC (131/140; 93.6%) compared to c4800-RP-GC (126/146; 86.3%), albeit not significantly (P = 0.6677). Of 9 samples testing positive by c6800 and negative by MP96-RP-GC, 7/9 (77.8%) were also negative by Xpert. By contrast, the number of samples returning a valid gyrA status was significantly (P = 0.0003) higher for MP96-RP-GC (270/293; 92.2%) compared to c4800-RP-GC (245/298; 82.2%). The overall MP96-RP-GC gyrA status correlated 98.4% (61/62) with the reported ciprofloxacin sensitive (35/36; 97.2%) or non-susceptible (26/26; 100%) phenotype. Improved RP-GC confirmatory rates and reported gyrA status were observed using MP96 nucleic acids compared to c4800 extracts. The data further highlight the ongoing need for NG supplemental testing for oropharyngeal samples.

Investigating oral human papillomavirus co-infection with Neisseria gonorrhoeae and Chlamydia trachomatis.

Compared to cervical cancer, little is known about human papillomavirus (HPV)-driven oropharyngeal cancer and their cofactors. Here, we investigated potential associations between Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) with oral HPV and HPV persistence, which are known cofactors in cervical carcinogenesis, and also play a role in HPV-driven oropharyngeal cancer. Saliva samples (n = 547) from 312 people were tested for CT and NG and whom had previously been tested for oral HPV infection in a longitudinal study. Eight participants were positive for CT (2.6%) and one for NG (0.3%). Six of these nine participants were also positive for oral HPV in at least one of their samples. We found no significant associations between HPV, CT, or NG infection in the saliva samples analyzed. These preliminary data suggest CT and NG have little influence on oral HPV-positivity and persistence in a general population. However, larger studies focusing on 'at risk' population cohorts are necessary to assess potential associations between oral sexually transmissible infections and oral HPV infections, and their outcomes.

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.

Decreased Neisseria gonorrhoeae genotypic diversity following COVID-19 restrictions in Queensland, Australia 2020.

We investigated the potential effects of COVID-19 public health restrictions on the prevalence and distribution of Neisseria gonorrhoeae (NG) genotypes in our Queensland isolate population in the first half of the year 2020. A total of 763 NG isolates were genotyped to examine gonococcal strain distribution and prevalence for the first 6 months of 2020, with 1 January 2020 to 31 March 2020 classified as 'pre' COVID-19 restrictions (n = 463) and 1 April 2020 to 30 June 2020 classified as 'post' COVID-19 restrictions (n = 300). Genotypes most prevalent 'pre' restrictions remained proportionally high 'post' restrictions, with some significantly increasing 'post' restrictions. However, genotype diversity was significantly reduced 'post' restrictions. Overall, it seems public health restrictions (9-10 weeks) were not sufficient to affect rates of infection or reduce the prevalence of well-established genotypes in our population, potentially due to reduced access to services or health-seeking behaviours.

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.

The changing epidemiology of Neisseria gonorrhoeae genogroups and antimicrobial resistance in Queensland, Australia, 2010-15: a case series analysis of unique Neisseria gonorrhoeae isolates.

BACKGROUND: Neisseria gonorrhoeae (NG) can lead to serious reproductive and sexual health outcomes, and the annual number of NG notifications in Australia increased steadily from 10329 in 2010 to 29549 by 2020. Australian populations most affected are urban men who have sex with men and First Nations peoples living in remote areas, and a resurgence in urban heterosexuals has been observed since 2012. METHODS: A case series analysis of Queensland NG isolates (2010-15) exploring temporal trends and antimicrobial resistance by demographic and geographic distribution and genotype was performed. Proportions describe age, sex, strain, genogroup (NG multi-antigen sequence typing), region, swab site, antimicrobial sensitivity and isolate rates per 100000 population. Dominant genogroups were identified. RESULTS: Among 3953 isolates, the median age was 25years (IQR 20-34years) and most (n =2871/3915, 73%) were men. Brisbane city (68.8) and Far North Queensland (54.1) excluding Cairns showed the highest rates. Forty-six genogroups were documented, seven (G2992, G6876, G1415, G4186, G5, G1407 and G6937) comprised half of all isolates. The predominant male genogroup was G2992 (16%), and G6876 (20%) for females; G5 was predominantly male from 2010 to 2011, but equal in both sexes from 2012 to 2015. CONCLUSION: Considerable temporal, geographical and demographical diversity was observed in Queensland NG isolates, which has public health implications. Certain genogroups are more transient than others, and evidence suggests bridging from male-dominant networks to heterosexual networks. Molecular surveillance can enhance tracking the epidemiology and movement of NG in Australia, highlighting the necessity of genotyping to expose potentially prevalent strains circulating in undetected or underrepresented networks by current screening methods.

A Gonococcal Vaccine Has the Potential to Rapidly Reduce the Incidence of Neisseria gonorrhoeae Infection Among Urban Men Who Have Sex With Men.

BACKGROUND: A gonococcal vaccine is urgently needed due to increasing gonorrhea incidence and emerging multidrug-resistant gonococcal strains worldwide. Men who have sex with men (MSM) have among the highest incidences of gonorrhea and may be a key target population for vaccination when available. METHODS: An individual-based, anatomical site-specific mathematical model was used to simulate Neisseria gonorrhoeae transmission in a population of 10 000 MSM. The impact of vaccination on gonorrhea prevalence was assessed. RESULTS: With a gonococcal vaccine of 100% or 50% protective efficacy, gonorrhea prevalence could be reduced by 94% or 62%, respectively, within 2 years if 30% of MSM are vaccinated on presentation for sexually transmitted infection (STI) testing. Elimination of gonorrhea is possible within 8 years with vaccines of ≥ 50% efficacy lasting 2 years, providing a booster vaccination is available every 3 years on average. A vaccine's impact may be reduced if it is not effective at all anatomical sites. CONCLUSIONS: Our study indicates that with a vaccine of modest efficacy and an immunization strategy that targets MSM presenting for STI screening, the prevalence of gonorrhea in this population could be rapidly and substantially reduced.

Combination Therapy for Mycoplasma genitalium, and New Insights Into the Utility of parC Mutant Detection to Improve Cure.

BACKGROUND: Mycoplasma genitalium (MG) infection is challenging to cure because of rising antimicrobial resistance and limited treatment options. METHODS: This was a prospective evaluation of the efficacy and tolerability of resistance-guided combination antimicrobial therapy for MG treatment at Melbourne Sexual Health Centre (August 2019-December 2020). All patients received 7 days of doxycycline before combination therapy based on the macrolide-resistant profile. Macrolide-susceptible infections received combination doxycycline + azithromycin (1 g, day 1; 500 mg, days 2-4) and macrolide-resistant infections combination doxycycline + moxifloxacin (400 mg daily for 7 days). Adherence and adverse effects were recorded at test-of-cure, recommended 14-28 days after antimicrobial completion. Sequencing was performed to determine the prevalence of single nucleotide polymorphisms (SNPs) in the parC gene and their association with moxifloxacin treatment outcomes in macrolide-resistant infections. RESULTS: Of 100 patients with macrolide-susceptible MG treated with doxycycline + azithromycin, 93 were cured (93.0%; 95% confidence interval [CI], 86.1-97.1). Of 247 patients with macrolide-resistant MG receiving doxycycline + moxifloxacin, 210 were cured (85.0%; 95% CI, 80.0-89.2). parC sequencing was available for 164 (66%) macrolide-resistant infections; 29% had SNPs at parC S83 or D87 (23% S83I). The absence of SNPs at parC S83/D87 was associated with 98.3% cure (95% CI, 93.9-99.8) following doxycycline + moxifloxacin. The presence of the parC S83I-SNP was associated with failure in 62.5% (95% CI, 45.8-77.3). Side effects were common (40%-46%) and predominantly mild and gastrointestinal. CONCLUSIONS: Combination doxycycline + azithromycin achieved high cure for macrolide-susceptible infections. However, in the context of a high prevalence of the parC S83I mutation (23%) in macrolide-resistant infections, doxycycline + moxifloxacin cured only 85%. Infections that were wild-type for S83/D87 experienced high cure following doxycycline + moxifloxacin, supporting the use of a parC-resistance/susceptibility testing strategy in clinical care.

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.

Optimized Method for Bacterial Nucleic Acid Extraction from Positive Blood Culture Broth for Whole-Genome Sequencing, Resistance Phenotype Prediction, and Downstream Molecular Applications.

The application of direct metagenomic sequencing from positive blood culture broth may solve the challenges of sequencing from low-bacterial-load blood samples in patients with sepsis. Forty prospectively collected blood culture broth samples growing Gram-negative bacteria were extracted using commercially available kits to achieve high-quality DNA. Species identification via metagenomic sequencing and susceptibility prediction via a machine-learning algorithm (AREScloud) were compared to conventional methods and other rapid diagnostic platforms (Accelerate Pheno and blood culture identification [BCID] panel). A two-kit method (using MolYsis Basic and Qiagen DNeasy UltraClean kits) resulted in optimal extractions. Taxonomic profiling by direct metagenomic sequencing matched conventional identification in 38/40 (95%) samples. In two polymicrobial samples, a second organism was missed by sequencing. Prediction models were able to accurately infer susceptibility profiles for 6 common pathogens against 17 antibiotics, with an overall categorical agreement (CA) of 95% (increasing to >95% for 5/6 of the most common pathogens, if Klebsiella oxytoca was excluded). The performance of whole-genome sequencing (WGS)-antimicrobial susceptibility testing (AST) was suboptimal for uncommon pathogens (e.g., Elizabethkingia) and some ß-lactamase inhibitor antibiotics (e.g., ticarcillin-clavulanate). The time to pathogen identification was the fastest with BCID (1 h from blood culture positivity). Accelerate Pheno provided a susceptibility result in approximately 8 h. Illumina-based direct sequencing methods provided results in time frames similar to those of conventional culture-based methods. Direct metagenomic sequencing from blood cultures for pathogen detection and susceptibility prediction is feasible. Additional work is required to optimize algorithms for uncommon species and complex resistance genotypes as well as to streamline methods to provide more rapid results.

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.

Proof-of-concept, rapid, instrument-free molecular detection of Neisseria gonorrhoeae and ciprofloxacin susceptibility.

OBJECTIVES: To develop instrument-free point-of-care methods using recombinase polymerase amplification (RPA) technology coupled with a simple lateral flow detection system to detect Neisseria gonorrhoeae and susceptibility to ciprofloxacin. METHODS: For identification of gonococcal infection, an RPA-based method was developed targeting the gonococcal porA pseudogene (NG-porA-RPA). For ciprofloxacin susceptibility, predictive WT sequences at codons 91 and 95 of the gonococcal gyrA DNase gene were targeted. Given the known complexities of SNP detection using RPA (e.g. the ability to accommodate mismatches) we trialled several different assays incorporating various additional non-template mismatches in the oligonucleotide sequences to reduce affinity for the mutant (resistant) gyrA sequences. Assays were evaluated using a bank of N. gonorrhoeae (n = 10) and non-gonococcal (n = 5) isolates and a panel of N. gonorrhoeae nucleic acid amplification test (NAAT)-positive clinical sample extracts (n = 40). RESULTS: The NG-porA-RPA assay was specific to N. gonorrhoeae and provided a positive percentage agreement (PPA) of 87.5% (35/40) compared with a commercial N. gonorrhoeae NAAT when applied to the 40 clinical sample extracts. For gyrA, the non-template bases successfully reduced banding intensity for double-mutant strains (mutations at both 91 and 95), but not for rarer single-mutant (91 only) strains. The most promising gyrA assay, NG-gyrA-RPA08, correctly detected 83% (25/30) of infections from NAAT-positive clinical samples confirmed to have WT gyrA sequences based on Sanger sequencing. CONCLUSIONS: These proof-of-concept data show that RPA technology has considerable promise for detecting N. gonorrhoeae and associated antibiotic susceptibility and would offer a diagnostic-based stewardship strategy identified as urgently needed by the WHO.

Modelling response strategies for controlling gonorrhoea outbreaks in men who have sex with men in Australia.

The ability to treat gonorrhoea with current first-line drugs is threatened by the global spread of extensively drug resistant (XDR) Neisseria gonorrhoeae (NG) strains. In Australia, urban transmission is high among men who have sex with men (MSM) and importation of an XDR NG strain in this population could result in an epidemic that would be difficult and costly to control. An individual-based, anatomical site-specific mathematical model of NG transmission among Australian MSM was developed and used to evaluate the potential for elimination of an imported NG strain under a range of case-based and population-based test-and-treat strategies. When initiated upon detection of the imported strain, these strategies enhance the probability of elimination and reduce the outbreak size compared with current practice (current testing levels and no contact tracing). The most effective strategies combine testing targeted at regular and casual partners with increased rates of population testing. However, even with the most effective strategies, outbreaks can persist for up to 2 years post-detection. Our simulations suggest that local elimination of imported NG strains can be achieved with high probability using combined case-based and population-based test-and-treat strategies. These strategies may be an effective means of preserving current treatments in the event of wider XDR NG emergence.

Surveillance systems to monitor antimicrobial resistance in Neisseria gonorrhoeae: a global, systematic review, 1 January 2012 to 27 September 2020.

BackgroundEffective surveillance of antimicrobial resistance (AMR) in Neisseria gonorrhoeae is required for the early detection of resistant strains and to ensure that treatment guidelines are appropriate for the setting in which they are implemented. AMR in N. gonorrhoeae has been identified as a global health threat.AimWe performed a systematic review to identify and describe surveillance systems targeting AMR in N. gonorrhoeae.MethodsWe searched Medline, PubMed, Global Health, EMBASE, CINAHL, Web of Science and ProQuest databases and grey literature between 1 January 2012 and 27 September 2020. Surveillance systems were defined as the continuous, systematic collection, analysis and interpretation of N. gonorrhoeae resistance data. The key components of surveillance systems were extracted, categorised, described and summarised.ResultsWe found 40 publications reporting on N. gonorrhoeae AMR surveillance systems in 27 countries and 10 multi-country or global surveillance reports. The proportion of countries with surveillance systems in each of the WHO's six regions ranged from one of 22 countries in the Eastern Mediterranean and five of 54 in Africa, to three of 11 countries in South East Asia. Only four countries report systems which are both comprehensive and national. We found no evidence of a current surveillance system in at least 148 countries. Coverage, representativeness, volume, clinical specimen source, type and epidemiological information vary substantially and limit interpretability and comparability of surveillance data for public health action.ConclusionGlobally, surveillance for N. gonorrhoeae AMR is inadequate and leaves large populations vulnerable to a major public health threat.

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.

MicroPIPE: validating an end-to-end workflow for high-quality complete bacterial genome construction.

BACKGROUND: Oxford Nanopore Technology (ONT) long-read sequencing has become a popular platform for microbial researchers due to the accessibility and affordability of its devices. However, easy and automated construction of high-quality bacterial genomes using nanopore reads remains challenging. Here we aimed to create a reproducible end-to-end bacterial genome assembly pipeline using ONT in combination with Illumina sequencing. RESULTS: We evaluated the performance of several popular tools used during genome reconstruction, including base-calling, filtering, assembly, and polishing. We also assessed overall genome accuracy using ONT both natively and with Illumina. All steps were validated using the high-quality complete reference genome for the Escherichia coli sequence type (ST)131 strain EC958. Software chosen at each stage were incorporated into our final pipeline, MicroPIPE. Further validation of MicroPIPE was carried out using 11 additional ST131 E. coli isolates, which demonstrated that complete circularised chromosomes and plasmids could be achieved without manual intervention. Twelve publicly available Gram-negative and Gram-positive bacterial genomes (with available raw ONT data and matched complete genomes) were also assembled using MicroPIPE. We found that revised basecalling and updated assembly of the majority of these genomes resulted in improved accuracy compared to the current publicly available complete genomes. CONCLUSIONS: MicroPIPE is built in modules using Singularity container images and the bioinformatics workflow manager Nextflow, allowing changes and adjustments to be made in response to future tool development. Overall, MicroPIPE provides an easy-access, end-to-end solution for attaining high-quality bacterial genomes. MicroPIPE is available at https://github.com/BeatsonLab-MicrobialGenomics/micropipe .

Reflex Detection of Ciprofloxacin Resistance in Neisseria gonorrhoeae by Use of the SpeeDx ResistancePlus GC Assay.

Resistance-guided therapy (RGT) for gonorrhea may reduce unnecessary use of broad-spectrum antibiotics. When reflexed from the Aptima Combo 2 assay, the ResistancePlus GC assay demonstrated 94.8% sensitivity and 100.0% specificity for Neisseria gonorrhoeae detection. Of the 379 concordant N. gonorrhoeae-positive samples, 86.8% were found to possess the gyrA S91F mutation, which was highly predictive for ciprofloxacin resistance and stable across 3,144 publicly available N. gonorrhoeae genomes. Our work supports the feasibility of implementing RGT for gonorrhea into routine molecular workflows.

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.

Evaluation of the SpeeDx ResistancePlus® GC and SpeeDx GC 23S 2611 (beta) molecular assays for prediction of antimicrobial resistance/susceptibility to ciprofloxacin and azithromycin in Neisseria gonorrhoeae.

BACKGROUND: Accurate molecular assays for prediction of antimicrobial resistance (AMR)/susceptibility in Neisseria gonorrhoeae (Ng) can offer individualized treatment of gonorrhoea and enhanced AMR surveillance. OBJECTIVES: We evaluated the new ResistancePlus® GC assay and the GC 23S 2611 (beta) assay (SpeeDx), for prediction of resistance/susceptibility to ciprofloxacin and azithromycin, respectively. METHODS: Nine hundred and sixty-seven whole-genome-sequenced Ng isolates from 20 European countries, 143 Ng-positive (37 with paired Ng isolates) and 167 Ng-negative clinical Aptima Combo 2 (AC2) samples, and 143 non-gonococcal Neisseria isolates and closely related species were examined with both SpeeDx assays. RESULTS: The sensitivity and specificity of the ResistancePlus® GC assay to detect Ng in AC2 samples were 98.6% and 100%, respectively. ResistancePlus® GC showed 100% sensitivity and specificity for GyrA S91 WT/S91F detection and 99.8% sensitivity and specificity in predicting phenotypic ciprofloxacin resistance. The sensitivity and specificity of the GC 23S 2611 (beta) assay for Ng detection in AC2 samples were 95.8% and 100%, respectively. GC 23S 2611 (beta) showed 100% sensitivity and 99.9% specificity for 23S rRNA C2611 WT/C2611T detection and 64.3% sensitivity and 99.9% specificity for predicting phenotypic azithromycin resistance. Cross-reactions with non-gonococcal Neisseria species were observed with both assays, but the analysis software solved most cross-reactions. CONCLUSIONS: The new SpeeDx ResistancePlus® GC assay performed well in the detection of Ng and AMR determinants, especially in urogenital samples. The GC 23S 2611 (beta) assay performed relatively well, but its sensitivity, especially for predicting phenotypic azithromycin resistance, was suboptimal and further optimizations are required, including detection of additional macrolide resistance determinant(s).

Analytical validation of a real-time hydrolysis probe PCR assay for quantifying Plasmodium falciparum parasites in experimentally infected human adults.

BACKGROUND: Volunteer infection studies have become a standard model for evaluating drug efficacy against Plasmodium infections. Molecular techniques such as qPCR are used in these studies due to their ability to provide robust and accurate estimates of parasitaemia at increased sensitivity compared to microscopy. The validity and reliability of assays need to be ensured when used to evaluate the efficacy of candidate drugs in clinical trials. METHODS: A previously described 18S rRNA gene qPCR assay for quantifying Plasmodium falciparum in blood samples was evaluated. Assay performance characteristics including analytical sensitivity, reportable range, precision, accuracy and specificity were assessed using experimental data and data compiled from phase 1 volunteer infection studies conducted between 2013 and 2019. Guidelines for validation of laboratory-developed molecular assays were followed. RESULTS: The reportable range was 1.50 to 6.50 log10 parasites/mL with a limit of detection of 2.045 log10 parasites/mL of whole blood based on a parasite diluted standard series over this range. The assay was highly reproducible with minimal intra-assay (SD = 0.456 quantification cycle (Cq) units [0.137 log10 parasites/mL] over 21 replicates) and inter-assay (SD = 0.604 Cq units [0.182 log10 parasites/mL] over 786 qPCR runs) variability. Through an external quality assurance program, the QIMR assay was shown to generate accurate results (quantitative bias + 0.019 log10 parasites/mL against nominal values). Specificity was 100% after assessing 164 parasite-free human blood samples. CONCLUSIONS: The 18S rRNA gene qPCR assay is specific and highly reproducible and can provide reliable and accurate parasite quantification. The assay is considered fit for use in evaluating drug efficacy in malaria clinical trials.